Figure 1.
Schematic of the AUTS2 genomic region.
Human accelerated sequences are shown as blue lines above the gene [17]–[19]. Structural variants [4]–[12], [14], [15] are represented as colored lines (red: deletion, orange: inversion, green: duplication, purple: translocation). The rs6943555 SNP associated with alcohol consumption [16] is shown as a magenta star. Arrows in bars signify that the structural variant extends past the gene in that direction. Exons are depicted as light blue rectangles, as defined by the RefSeq genes track in the UCSC Genome Browser [52]. Numbers to the left of the lines correspond to a reference number. Human Accelerated Conserved Non-coding Sequence (HACNS), Human Accelerated Region (HAR), developmental delay (DD), intellectual disability (ID), dysmorphic features (DF), seizure disorder (SD), multiple congenital anomalies (MCA), language disability (LD).
Figure 2.
auts2 48 hpf morphant phenotype.
(A) Fish injected with the 5 base-pair translational MO mismatch control have similar morphology as wild type fish. Injection of the auts2 translational MO results in fish with a stunted development phenotype that includes a smaller head, eyes, body and fins. (B) HuC-GFP fish injected with the 5 bp control MO display normal levels of developing neurons in the brain. HuC-GFP translational MO injected fish display considerably less developing neurons in the optic tectum (ot), retina (ret), and cerebellum (ce). (C) 5 bp mismatch control injected fish have little to non-observable apoptosis in the brain as observed by TUNEL staining, while translational MO injected fish display high levels of apoptosis, primarily in the midbrain (mb) and hindbrain (hb). (D) PCNA cell proliferation assay in the 5 bp MO control injected fish shows lower levels of cell proliferation in the brain compared to the translational MO injected fish. (E) Tg(mnx1∶GFP) fish injected with the 5 bp MO control display normal levels of motor neurons versus the auts2 translational MO injected fish which have fewer motor neurons in the spinal cord (sc). In addition, motor neuron projections (mnp) are weaker and more perpendicular to the spinal cord. (F) Translational MO injected fish display fewer Rohon-Beard cells (arrowheads) in the spinal cord than morphants. All morphant fish are scaled to their 5 bp control counterparts.
Figure 3.
Enhancers within an ASD–associated AUTS2 intronic deletion [5].
Three positive enhancers (AEC27, 29, 32) show positive enhancer activity in zebrafish (24 or 48 hpf) and in mice (E11.5 or 12.5). AEC27 shows enhancer expression in the somitic muscle in zebrafish, while in mouse at E11.5 (hs658; [34]) it is active in the midbrain, medulla, and neural tube at E11.5. The histological section below shows its enhancer activity in the pretectum and the pons. At E12.5, AEC29 shows enhancer activity in the olfactory epithelium (arrows in histological section) similar to zebrafish and in addition also displays enhancer expression in the eye. AEC32 recapitulates the zebrafish enhancer expression displaying strong enhancer activity in the midbrain (tectum) and hindbrain and in addition also displays enhancer expression in the forebrain at E12.5. Histological sections of AEC32 show enhancer activity in the mouse cerebellum (red arrowheads).
Figure 4.
Four positive zebrafish and mouse enhancers in regions implicated in human evolution.
At E12.5, AEC10 shows zebrafish and mouse enhancer expression in the midbrain and eye. The histological section below highlights its expression in the tectum. AEC13, is expressed in the otic vesicle both in zebrafish and E11.5 mouse embryos (hs1660 ; [34]). AEC21 is expressed in the spinal cord in zebrafish, while in the mouse it showed midbrain expression at E11.5 (hs1425; [34]). Histological sections below show its expression in the pretectum of the midbrain. AEC24 was expressed in the spinal cord and hindbrain in zebrafish and in the eye in mouse at E12.5.