|
Fig. 1 – RAI1/Rai1 protein organization and induction by retinoic acid. (A) A schematic representation of the Rai1 protein in human, mouse, two frog species (X. laevis and X. tropicalis), and zebrafish. This illustrates that the organization of the primary protein domains, including the Trans-Activation Domain (TAD), Nuclear Localization Signal containing region (NLS; see Supplementary material Fig. S1B for details), and extended plant homeodomain (ePHD/ADD, also known as a PHD finger) are conserved in the species examined (see Supplementary material Fig. S1A for sequence alignment of the PHD finger). A 14 poly-glutamine stretch (Q) is found in humans and reduced glutamine stretches can also be found in a similar region of other species except zebrafish. The NLS region contains the first known nuclear localization sequence (Darvekar et al., 2013). Figure not to scale. (B) Retinoic acid induces expression of rai1 during Xenopus development. Quantitative PCR of rai1 mRNA after all-trans retinoic treatment (ATRA) at stage 23 showing significant 3.91-fold increase (t-test, p = 0.0037) relative to the control housekeeping gene (ef1 alpha). (C) Retinoic acid may directly regulate rai1. A histogram of RAR binding sites in a 2 kb region upstream of the rai1 start site in X. laevis reveals the enrichment of retinoic acid receptor binding sites.
|
|
Fig. 2 Expression and loss of function reveal a role for Rai1 in craniofacial and brain development. (A) In situ hybridization of rai1 mRNA in Xenopus tropicalis. (i) Dorsolateral view at stage 18–19 revealing labeling throughout the developing nervous system. (ii) Lateral view of stage 23, revealing rai1 in the presumptive migrating neural crest (arrows) and eye. (iii) Lateral view of stage 26 revealing rai1 in the presumptive migrating neural crest (arrows). (iv) Lateral view of stage 33 revealing rai1 in the branchial arches (arrows) eye, optic vesicle (ov) and muscle segments (ms). (v) Dorsal view at stage 33 revealing rai1 in the central nervous system (CNS). (vi) Lateral view at stage 41 revealing rai1 throughout the head. (vii) Transverse sections through the head at stage 41 revealing that rai1 is primarily located outside the CNS in the tissues of the face at this stage. Abbreviations, np; neural plate, ms; muscle segments, ov; otic vesicle, cg; cement gland, CNS; central nervous system. (B) Decreased function of Rai1 results in dramatic effects on Xenopus laevis development including craniofacial defects. (i) Lateral view of representative embryo at stage 42 injected with control morpholino (MO). (ii) Lateral view of representative embryo at stage 42 injected with 5 ng of MO. Defects include digestive tract, shortened tail and smaller head. (iii) Lateral view of representative embryo at stage 42 injected with 10 ng of Rai1 MO. There are severe defects in digestive tract, tail and head development.
|
|
Fig 4. Rai1 is critical for neural crest migration and to establish correct cartilage size. Rai1 morphants injected with 5 ng of morpholino were used for analysis. (A) in situ hybridizations of the neural crest marker, ap-2 in control (i, ii) and Rai1 morphants (iii, iv), showing an aberrant pattern of neural crest during early migration (stage 23). Arrows indicate position of the developing embryonic mouth for context. (B) in situ hybridizations of ap-2 in control (i, ii) and Rai1 morphants (iii, iv), showing an aberrant pattern of neural crest after migration (stage 30). Arrows indicate position of the developing embryonic mouth for context. (C) Cartilage labeling by Alcian Blue in control (i, ii) and Rai1 morphants (iii, iv) illustrates a decreased size of cartilage elements at stage 45. Abbreviations: eth; ethmoid, nc; nasal capsule, MK; Meckel’s, ir; infrarostral, ch; ceratohyal, ba; branchial, bh; basohyal.
|
|
Fig. 5 – Decreased Rai1 results in defects in axon patterns, brain morphology, increased forebrain apoptosis and decreased bdnf expression. Rai1 morphants injected with 5 ng of morpholino were used for each analysis. (A, B) Dorsal view of representative tadpoles (stage 42) labeled with a neurofilament marker (3A10) showing aberrant axon patterns and axonal beading in Rai1 morphants (arrowheads). The brain is outlined with white dots to provide context. (C, D) Dorsal view of representative tadpoles (stage 42) where the ventricles were injected with Texas Red labeled dextran. Fluorescent images were superimposed onto the light micrographs to provide context. Results show that Rai1 morphants have dramatically decreased ventricle space in the forebrain. (E, F) A marker of apoptosis, cleaved caspase-3 (green) reveals increased cell death in the forebrain of Rai1 morphants. Shows a compressed z-stack of a midline sagital section at stage 33–34. Counterstained with nuclear marker (propidium iodide; red). The brain is outlined with white dots and the position of the developing embryonic mouth is indicated by a white arrow for context. (G) Bar graph of the average number of cleaved caspase-3 positive cells showing a significant difference (t tests, brain; p = 2.48E-10, face; p = 0.713) in the brain (star) but not in the face. (H) qPCR analysis of bdnf expression in Rai1 morphants at stage 25–26 (28 hpf). Expression was normalized to ef1alpha and presented as a fold change compared to control. Results indicate a statistically significant negative 1.5-fold change (t test, p = 0.0049) in Rai1 morphants.
|
|
Fig. 3 – Rai1 is required for normal orofacial development. Rai1 morphants injected with 5 ng of morpholino were analyzed at stage 42. (A) Representative frontal views (i, iii) and lateral views (ii, iv) of embryos injected with 5 ng of Rai1 and control MO. The mouth is outlined in red dots (i and iii) or indicated with a red arrow (ii and iv). In lateral views (ii and iv) the head is outlined with white dots to emphasize the protruding forehead. The snout appears shorter and the mouth is smaller and rounder in the Rai1 morphants than controls. Abbreviations: cg; cement gland. (B) Traditional measurements of facial dimensions in Rai1 morphants verses controls. Schematics indicate where measurements were taken. Data as bar graphs (i– v), show statistically a shorter snout, smaller midface area, a narrower face, and rounder mouth. (C) Morphometric analysis of orofacial shape in Rai1 morphants supports tradition measurements and provides details of changes in orofacial shape. (i) Principal component analysis shows distinct separation of orofacial shapes in Rai1 morphants and controls. Control morphants are in black and Rai1 morphants are in red. (ii) Discriminant function analysis showing the statistically significant procrustes distance and p-value as well as the transformation grid of the changes in landmark location. Note the dramatic shift in nostril landmarks (arrows) and corresponding warping in the midface indicative of midface hypoplasia. Also notable is the shifts in mouth landmarks consistent with a rounder shape mouth.
|
|
Supplementary Figure 2. A,B) Xenopus laevis in situ hybridization using a probe generated from the 3’UTR of the Xenopus tropicalis rai1 sequence. The region shares a 66% nucleotide similarity with the same region of the X. laevis rai1 gene and showed a highly similar staining pattern in X. laevis embryos. The probe sequence did not match any other sequence in either Xenopus species with identity of greater than 20% or with a stretch of nucleotides longer than 50. C,D) The sense or negative control probe show no significant staining pattern. A blue background was necessary to show contrast with the starkly white embryos.
|
|
Supplemental Figure S3) Establishing Rai1 morpholino specificity. A) A second translation blocking morpholino (Rai1 MO2) results in very similar phenotypes as the first morpholino used in this study. (i) Lateral view of representative embryo at stage 42 injected with control morpholino (Cont MO). (ii) Lateral view of three representative embryos at stage 42 injected with 5ng of Rai1 MO2. Defects include malformed digestive tract, shortened tail and smaller head. (iii) Lateral view of representative embryo at stage 42 injected with 10ng of Rai1 MO. There are more severe defects in the digestive tract, tail and head. (iv) Frontal view of the face of a control embryo at stage 42. (v) Frontal view of the face of an embryo injected with 5ng of Rai1 MO2. The face is narrower and the mouth rounder as in embryos injected with the same amount of original Rai1 morpholino. B) (iv) Transverse section of stage 20 embryo injected with control MO and labeled with antibodies to human RAI1. (v) Transverse section of stage 20 embryo injected with Rai1 MO and labeled with human Rai1 antibodies revealing a noticeable decrease in immunofluorescence. (vi) Quantification of Rai1 immunofluorescence by pixel intensity using photoshop software. Results show a 2.16 fold decrease that is significant (t-test, p=0.000116, n= 10). Abbreviations, np; neural plate, nc; notochord, cg; cement gland.
Note in this and supplemental methods-
The pixel intensity from 10 embryos from the two experiments was pooled and averaged.
|
|
Supplemental Figure S4. in situ hybridizations of ap-2 (a neural crest marker) in embryos injected into one cell at the two cell stage. These embryos have morpholino in one bilateral half of the body. Control embryos were not different than controls injected at the one cell stage and therefore is not shown. A) Shows the uninjected side displaying normal ap-2 expression pattern. B) Shows the injected side in the same embryo as A. ap-2 expression is clearly aberrant. C) Shows a frontal view of the same embryo from A and B. The arrow is pointing to the injected side.
|
|
rai1 (retinoic acid induced 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 18, dorso-lateral view, anterior left.
|
|
rai1 (retinoic acid induced 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
|
|
|
|
rai1 (retinoic acid induced 1) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, lateral view, anterior left, dorsal up.
|
|
|
|
rai1 (retinoic acid induced 1) gene expression in Xenopus laevis embryo, head and trunk region only, assayed via in situ hybridization, NF stage 28, lateral view, anterior left, dorsal up (image on left); and dorsal view anterior left, dorsal up ( image on right).
|
