Article Figures & Data
- Figure 1.
Displacement of cranial DRG and loss of peripheral sensory projections in Brn3a/Islet1 DKO embryos. A–D, Immunofluorescence staining for Brn3a and Islet1 expression in the cervical DRG of E12.5 embryos. Wnt1-Cre produces nearly complete excision of Islet1 in the DRG but spinal motor neuron expression is unaffected. E, Distance from dorsal margin of DRG to dorsal margin of spinal cord in control and DKO embryos, showing dorsal displacement of DRG in DKO embryos. F, E12.5 control embryo stained for βgalactosidase activity and imaged using OPT. G–J, Close-up view of the trigeminal system in four genotypes. All branches are diminished and the mandibular branch is nearly absent (arrow, J) in the DKO embryo. An oblique view is shown to avoid the superimposition of bilateral structures. K–N, Close-up of the cervical plexus derived from C2–C5. Lateral view. O–R, Close-up of the brachial plexus and innervation of the forelimb, derived from C5–T1 (posterior view). MN, Motor neurons; BP, brachial plexus; CP, cervical plexus; Op, ophthalmic trigeminal; Mx, maxillary trigeminal branch; Mn, mandibular trigeminal branch; R, radial nerve; M, median nerve; U, ulnar nerve.
- Figure 2.
The Brn3a/Islet1 compound sensory phenotype at mid-gestation. The profound defects in peripheral innervation noted at E12.5 persist at E14.5 in the DRG (A–D) and the TG (E–H). The size of the ganglia are markedly reduced in Islet1 CKO embryos. In DKO embryos, the DRG are displaced dorsally and the dorsal roots are markedly foreshortened (arrows, D). In DKO embryos, the cervical plexus, brachial plexus, and mandibular branch of the trigeminal nerve (arrow in E–H) are nearly absent. 8g, Vestibulocochlear ganglion; 9g, superior ganglion; C2, cervical DRG 2; T1, thoracic DRG 1; BP, brachial plexus; CP, cervical plexus; Hb, hindbrain; Mn, mandibular branch trigeminal nerve; TG, trigeminal ganglion.
- Figure 3.
Loss of subtype specification in Brn3a/Islet1 DKO embryos. A, Markers of sensory subtypes were examined by immunofluorescence in matched sagittal sections of cervical DRGs from Brn3a KO, Islet1 CKO, DKO, and control E13.5 embryos. B, Key markers of sensory subtype differentiation were examined in the TG of control and DKO ganglia at E13.5. Horizontal sections are shown. TrkB expression is restricted to specific cells in control ganglia; TrkB expression persists in DKO ganglia but no longer identifies a specific set of cell bodies. Dashed lines indicate location of the DRG in sections in which the fluorescence signal cannot be distinguished from background.
- Figure 4.
Immunofluorescence and in situ hybridization of novel Brn3a/Islet1 DKO targets. Cervical DRG of E12.5 embryos were examined in single KO, DKO, and control embryos. Antisera for Islet2, Prrxl1, and Brn3b confirmed the loss of these transcription factors in DKO ganglia, while the marker of glial precursors Sox10 was unaffected. Dashed lines indicate the location of DRG in sections in which the signal cannot be clearly distinguished from background.
- Figure 5.
Analysis of epistasis between Brn3a and Islet1 mutations. Epistatic interactions between the Brn3a KO and Islet1 DKO genotypes were examined for 479 transcripts which are at least threefold changed relative to WT in at least one of the three mutant genotypes. A, Two-dimensional epistasis plot in which the x-axis represents the change in expression due to the loss of Brn3a function in the presence of Islet1, based on a the difference between the WT and Brn3a KO expression levels, normalized to the maximum expression in any genotype: (EWT − EB3aKO)/max. The y-axis represents the change in expression due to the loss of Brn3a in the absence of Islet1, based on the difference between the Islet1 CKO and DKO expression levels: (EIsl1CKO − EDKO)/max. Transcripts for which the effects of the Brn3a KO and Islet1 CKO genotypes are additive appear on the diagonal. Transcripts showing negative epistasis are shifted to the left and right of the diagonal, and those showing positive epistasis are shifted above and below the diagonal. B, Individual regulated transcripts exhibit negative epistasis, additive effects, and positive epistasis between the Brn3a KO and Islet1 CKO genotypes. C, Distribution plots of difference values for comparisons between WT and DKO ganglia for the most-changed 479 transcripts and a control set of randomly selected transcripts. D, Histogram plots of distributions of (EWT − EB3aKO)/max and (EIsl1CKO − EDKO)/max. E, Distributions of (EWT − EIsl1KO)/max and (EBrn3aCKO − EDKO)/max. Comparisons of WT to single knock-outs exhibit broader distributions than comparisons between single knock-outs and DKO ganglia, confirming the predominant effect of global negative epistasis. p values are derived from K–S tests and represent the probability of observing the null hypothesis that the distributions do not differ significantly.
- Figure 6.
Chromatin immunoprecipitation of Islet1 bound to the Neurod4 locus. A–D, In situ hybridization for Neurod4 mRNA in the E13.5 TG. The normal developmental decline in expression observed in the WT TG fails to occur in Brn3a KO, Islet1 CKO or DKO ganglia. E, ChIP was performed using formalin-fixed E11.5 TG and a polyclonal Islet1 antibody (see Materials and Methods). Quantitative PCR was performed using oligonucleotide primer sets spaced at intervals of 0.5–1 kb across the locus (Lanier et al., 2007), and enrichment of the selected over an unselected control sample was analyzed by the cycle threshold method (Livak and Schmittgen, 2001). The fold enrichment relative to input chromatin was normalized to the average of five control sites in the Alb1 (serum albumin) locus, which is not expressed in sensory neurons, as shown by the yellow line (1-fold enrichment). Islet1 ChIP showed two regions of specific selection: in the 5′ UTR −14 to −13 kb upstream and in the single intron 3.2–3.8 kb downstream from the start of transcription (region 1 and region 2, designated by red bars). Mean folds of two selections for each primer pair are shown. The enrichment maxima occurred at −13,397 and +3888. T tests using 3–6 PCR primer pairs and two independent selections were used to assess the significance of enrichment for these regions compared to a control region 3′ to the transcription unit (region 3). Region 1 versus region 3 enrichment, p = 2.4 × 10−6; region 2 versus region 3 enrichment, p = 4.5 × 10−5. Region 3 versus alb (albumin) locus, p = 0.85, 0.94 (n.s.). The regions of selection by Islet1 correspond closely to conserved enhancer elements containing known Brn3a binding sites (Lanier et al., 2007).
Gene symbol Gene title Fold Change Islet1/WT Brn3a/WT DKO/WT Hoxc9 Homeobox C9 20.4 3.8 25.7 Gnas Guanine nucleotide binding protein, α stimulating 0.9 0.7 23.7 Eya1 Eyes absent 1 9.4 7.8a 13.9 Lhx1 LIM homeobox protein 1 4.5c 0.7 13.6 Actc1 Actin, α, cardiac muscle 1 6.8 3.1 13.5 Dmrta2 Doublesex and mab-3 related txn factor like A2 3.1 2.2 10.8 Nova1 Neurooncological ventral antigen 1 4.0 1.2 10.7 Lhx2 LIM homeobox protein 2 11.7c 0.4 10.3 Irx3 Iroquois related homeobox 3 1.4 4.0 9.8 Lilrb3 Leukocyte immunoglobulin-like receptor, subfamily B 6.3 2.4 9.5 Myl1 Myosin, light polypeptide 1 24.8 8.3 9.5 Cartpt CART prepropeptide 4.7 11.3 9.3 Dlg2 Discs, large homolog 2 2.5 2.8 9.0 Gbx2 Gastrulation brain homeobox 2 1.0 0.6 8.7 Npff Neuropeptide FF-amide peptide precursor 3.1 1.5 8.7 Npy Neuropeptide Y 3.9 1.9 8.4 Lbxcor1 Ladybird homeobox corepressor 1 7.4c 0.7 8.4 Nxph4 Neurexophilin 4 2.8 0.8 8.3 Irx5 Iroquois related homeobox 5 1.5 3.8 8.1 Dcc Deleted in colorectal carcinoma 6.1 2.9a 8.0 Sall3 Sal-like 3 4.9 0.2 7.9 Clic6 Chloride intracellular channel 6 1.9 1.0 7.6 Hbb-bh1 Hemoglobin Z, embryonic 5.8 6.3 7.5 Crabp1 Cellular retinoic acid binding protein I 7.5 1.6 7.4 Irs4 Insulin receptor substrate 4 1.1 2.0 7.4 Mab21l1 Mab-21-like 1 4.7 5.4 7.3 Rbm24 RNA binding motif protein 24 9.5 1.4 7.1 Hoxc8 Homeobox C8 3.9 2.0 7.1 Vstm2a V-set and transmembrane domain containing 2A 2.3 1.1 7.1 Calb2 Calbindin 2; calretinin 0.4 1.3a 7.0 Hoxb5 Homeobox B5 3.2 3.3 6.8 Olig1 Oligodendrocyte txn factor 1 4.2 1.4 6.6 Ajap1 Adherens junction associated protein 1 4.2 7.4 6.6 Dab1 Disabled homolog 1 2.4 3.0 6.4 Transcripts with increased expression in Brn3a/Islet1 double knock-out DRG. Increased transcripts are ranked by the fold change in E12.5 DKO DRG relative to a wild-type control. Fold changes are derived from the averages of two biologically independent microarray assays. Numbers in bold indicate replicated significant change p values (p < 0.002, p > 0.998) for the comparison between the stated genotype and control ganglia. Some of the observed changes have been previously confirmed by in situ hybridization, immunofluorescence, or qPCR in the (TG) (a) or DRG (b) of Brn3a KO embryos or the DRG (c) of Islet1 CKO embryos (Eng et al., 2004, 2007; Sun et al., 2007; Lanier et al., 2009; Dykes et al., 2010).
Gene symbol Gene title Fold change WT/Islet1 WT/Brn3a WT/DKO Avil Advillin 6.3 14.3a 503 Speer1-ps1 Spermatogenesis associated E-rich 1, ps1 5.8 1.8 159 Fam19a4 Tafa4 1.8 8.7 90.6 Bnc1 Basonuclin 1 21.4 15.9b 71.1 Etv4 Ets variant gene 4 8.8 1.8 57.3 Pou4f1 POU domain, class 4, txn factor 1; Brn3a 1.0 42.7 50.9 Cckar Cholecystokinin A receptor 27.6 0.9 46.7 Gal Galanin 13.6c 14.7a 39.0 Hoxd1 Homeobox D1 12.1 5.4a 31.7 Gpr64 G protein-coupled receptor 64 1.7 4.0b 26.7 Pou4f2 POU domain, class 4, txn factor 2; Brn3b 1.0 110a 26.4 Runx1 Runt related txn factor 1 22.9c 3.7a,b 26.3 Ppp1r1c Protein phosphatase 1, regulatory subunit 1C 1.6 1.2 26.2 Pou4f3 POU domain, class 4, txn factor 3; Brn3c 1.2 4.0 23.6 A530088H08Rik Pirt 4.7 7.3 21.3 Pappa2 Pappalysin 2 0.9 9.8 21.3 Higd1c HIG1 domain family, member 1C 4.5 3.2 20.5 Th Tyrosine hydroxylase 8.3c 1.9a 15.0 Selected Prrxl1 Paired related homeobox protein-like 1; Drg11 2.5 3.6 12.8 Isl2 Insulin related protein 2; Islet 2 3.4c 3.5 12.2 Scn7a Na + channel, voltage-gated, VII α; NAG 2.3 2.9 10.9 Fgf13 Fibroblast growth factor 13 3.3 3.6 10.6 Fam38b Piezo 2 4.5 3.2 10.6 Etv1 Ets variant gene 1; Er81 3.0 1.8 9.6 Ntrk3 Neurotrophic tyr kinase, receptor, type 3; TrkC 1.0 6.1a 9.2 Cntn2 Contactin 2; Tag1 3.8 3.8 9.0 Etv5 Ets variant gene 5; ERM 3.8 2.8 8.6 Adcyap1 Adenylate cyclase activating polypeptide 1; PACAP 0.9 5.0 8.4 Prph Peripherin 1.5 1.2 8.1 Trpv1 Transient receptor potential cation channel, subfamily V, member 1 7.3c 8.3 7.2 Ntrk1 Neurotrophic tyr kinase, receptor, type 1, TrkA 8.7c 1.8a 5.0 Runx3 Runt related txn factor 3 0.8 11.4a 4.2 Scn9a Na+ channel, voltage-gated IX α; Nav1.7 1.9 1.8 4.0 Transcripts with decreased expression in Brn3a/Islet1 double knockout DRG. For details, see Table 1.
- Movie 1.
OPT imaging of WT and Brn3a/Islet1 DKO embryos. Embryos were harvested at E12.5, genotyped, fixed, LacZ-stained, and cleared for OPT as described in Materials and Methods. OPT was used to generate 400 rotational images of each embryo, and 200 such images, representing 180° of rotation, were used to produce the video file. Arrowheads indicate the foreshortened dorsal roots observed in the DKO embryo. CPx, Cervical plexus; BPx, brachial plexus; HB, hindbrain; SC, spinal cord; DRG, dorsal root ganglia; TG, trigeminal ganglion; Mn, mandibular branch, trigeminal system; Mx, maxillary branch, trigeminal system (infraorbital nerve); 8g, vestibulocochlear ganglion; 9g, superior ganglion.
