A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs - PubMed (original) (raw)

. 2008 Nov;11(11):1283-93.

doi: 10.1038/nn.2209. Epub 2008 Oct 12.

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A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs

Yunfu Sun et al. Nat Neurosci. 2008 Nov.

Abstract

We used conditional knockout strategies in mice to determine the developmental events and gene expression program regulated by the LIM-homeodomain factor Islet1 in developing sensory neurons. Early development of the trigeminal and dorsal root ganglia was grossly normal in the absence of Islet1. From E12.5 onward, however, Isl1 mutant embryos showed a loss of the nociceptive markers TrkA and Runx1 and a near absence of cutaneous innervation. Proprioceptive neurons characterized by the expression of TrkC, Runx3 and Etv1 were relatively spared. Microarray analysis of Isl1 mutant ganglia revealed prolonged expression of developmental regulators that are normally restricted to early sensory neurogenesis and ectopic expression of transcription factors that are normally found in the CNS, but not in sensory ganglia. Later excision of Isl1 did not reactivate early genes, but resulted in decreased expression of transcripts related to specific sensory functions. Together these results establish a central role for Islet1 in the transition from sensory neurogenesis to subtype specification.

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Figures

Figure 1

Figure 1. Defective development of the DRG and spinal nerves in Islet1 conditional knockout mice

The sensory ganglia in control and CKO embryos were compared at E11.5 (A–C) and E14.5 (D,E, H–J). Presence of a conditional Rosa26-LacZ allele, activated by Wnt1-cre, allowed staining of sensory and autonomic ganglia and their axons in whole embryo preparations. In cranial regions, extensive staining was also seen in other neural crest-derived tissues. (A) LacZ staining of E11.5 embryos showing grossly normal morphology and normal-sized DRG in Islet1 CKO embryos at this stage. (B) Islet1 expression in differentiating neurons of E11.5 control ganglia. Islet1+ neurons no longer express Sox10, a marker of multipotent precursors and glia. (C) Normal initiation of Brn3a expression in E11.5 CKO embryos. Sox10 is characteristic of DRG precursors and is not co-expressed with Brn3a in either genotype. Occasional appearance of co-labeling is due to nuclear overlap. (D,E) Thoracic region of whole-mount Xgal stained, hemisected E14.5 embryos, showing marked reduction of the DRG in the CKO specimen. There is also profound reduction of the sympathetic chain ganglia (arrowheads). (F) Brn3a immunoreactivity as a measure of neuronal number in E11.5 and E14.5 DRG. Asterisks indicate statistical significance (t-test) for a given comparison. E11.5, p=0.38; E14.5, p <0.001. (G) Increased cell death in the TG and DRG of CKO embryos. Caspase-3 immunoreactive cells were counted in 6–10 slides for each sample. E11.5 TG, p<0.001; E12.5 TG, p<0.001; E12.5 DRG p=0.02. (H) The mid-thoracic body wall of a hemisected E14.5 embryo, showing βgalactosidase expression activated by Wnt1-cre in the sensory but not the motor component of the intercostal nerves. In CKO embryos, labeling of the intercostal nerves is diminished but detectable, however the cutaneous branches (arrows in top panel) are absent. The esophagus is prominent in the lower panel but is out of the plane of focus in the top panel. (I) Innervation of the distal forelimb and hindlimb at E14.5. In CKO embryos fine cutaneous sensory branches are lost throughout the limb (inset views). In the digits, labeled sensory axons persist only in a single fiber bundle in a corresponding position in digits 1, 2, and 5 of both the forelimb and hindlimb (arrowheads). (J) Immunofluorescence for TrkA and TrkC in sensory fiber bundles innervating digit 5. In control embryos, sensory fibers are immunoreactive for both TrkA and TrkC. In CKO embryos, the medial axon bundle adjacent to digit 4 is not labeled, and the persisting fiber bundle is immunoreactive for TrkC only (arrows). Legend: 1–5, digits 1–5; SC, spinal cord; T1, T6, thoracic dorsal root ganglion 1, 6. Error bars in all figures indicate mean +/− S.D.

Figure 2

Figure 2. Neurotrophin receptor expression in sensory ganglia lacking Islet1

(A–D) Expression of TrkA and TrkC in the brachial level DRG of control and CKO embryos of the specified developmental stages. TrkC expression is delayed until E12.5 in Islet1 CKO ganglia. However, there is a subsequent loss of TrkA+ neurons and relative sparing of TrkC+ cells at later stages. TrkA immunoreactivity is also diminished in sensory fibers in the dorsal root (arrows, B). (E–F) Cell counts for TrkA and TrkC immunoreactive neurons. For control versus CKO: TrkA E11.5 p=0.39; E14.5 p=0.0002; TrkC E12.5 p=0.24; E14.5 p=0.0002 (increase). (G–H) TrkB expression is markedly diminished from E12.5 onward in the DRG of Islet1 CKO embryos. (I–J) TrkA and TrkC expression in the spinal projections of sensory neurons at E14.5. Innervation of the superficial layers of the spinal cord by TrkA fibers is markedly reduced, and ectopic fibers are observed (arrows, J). TrkC immunoreactive fibers appear undiminished, and project ventrally in both control and mutant specimens (arrowheads, I, J). SC, spinal cord.

Figure 3

Figure 3. Expression of transcription factors regulating sensory subtype specification is altered in the DRG of Islet1 CKO embryos

(A,B) Runx1 expression in the brachial level DRG at E12.5 and E14.5, showing markedly diminished expression in CKO ganglia. (C,D). Runx1, Ret and TrkA expression in P1 DRG. Subsets of neurons expressing all combinations of these markers appear markedly diminished in CKO DRG. (E–G) Runx3 and Islet1 expression in the DRG of control and CKO embryos at E11.5. In control embryos, Runx3 immunoreactive neurons are a subset of Islet1+ cells. However, in the absence of Islet1, Runx3 expression is largely preserved. In G the number of Runx3 cells shows a modest decrease in CKO ganglia which did not reach statistical significance. (H,I) Runx3 and TrkC expression at E12.5 and E14.5. Neurons expressing these proprioceptive markers are relatively spared in Islet1 CKO ganglia, and account for an increasing fraction of the remaining DRG neurons as development progresses. (J,K) At E11.5, Islet1 is co-expressed with Etv1 in a subset of DRG neurons. At E14.5, Etv1+ neurons are relatively spared, and nearly all co-express TrkC, indicating that they are highly overlapping with the Runx3/TrkC population. (L) At 14.5, Islet1 is extensively co-expressed with Runx1 but is no longer co-expressed with Runx3 or Etv1. Mot, motor neurons; SC, spinal cord.

Figure 4

Figure 4. Islet1 regulates early and late programs of sensory gene expression

Microarray analysis of E12.5 DRG reveals coordinated changes in expression of gene families regulating specific phases of sensory development. (A) Factors associated with sensory subtype specification, including members of the Trk, Runx and Ets families, show profound decreases for genes associated with nociceptive neurons and little or no change for markers of proprioceptive neurons. (B) Neurogenic genes of the bHLH family, associated with early steps in sensory differentiation, are increased in Islet1 CKO DRG. (C) Gene expression changes for NeuroD4 and Insm1 are concordant in the DRG and TG. Asterisks indicate significant increase or decrease (change p <0.002 or >0.998) in two independent comparisons. Scale: 200µm.

Figure 5

Figure 5. Ectopic activation of spinal/hindbrain gene expression in Islet1 CKO sensory ganglia

(A) Microarray analysis of E12.5 DRG reveals abnormal expression of transcription factors usually associated with spinal neuron development (Lhx1, Lhx2, Olig1, Olig2, Lbxcor1), and decreased expression of Islet2. LIM-interacting proteins of the Ldb and LMO families, which are normally expressed in both sensory ganglia and spinal cord, show relatively modest changes. Asterisks indicate significant increase or decrease (change p <0.002 or >0.998) in two concordant comparisons. (B) In situ hybridization shows expression of increased transcription factors increased in Islet1 CKO embryos in the spinal cord and DRG. Islet1 CKO views are shown at higher magnification to reveal detail. (C) Concordant abnormal expression of CNS transcription factors in the E12.5 trigeminal ganglia. DRG, dorsal root ganglion; HB, hindbrain; SC, spinal cord; TG, trigeminal ganglion. Scale: B, 100µm; C, 200µm.

Figure 6

Figure 6. Late excision of Islet1 supports nociceptor survival but alters downstream gene expression

Islet1F/F and Islet1CreER/+ mice were interbred to produce Islet1F/+ control and Islet1F/CreER IKO embryos. Tamoxifen was administered to pregnant animals at E11.5 and embryos were examined at E14.5 or E18.5. Because of the need to assess the extent of Islet1 knockdown in each experiment, in E14.5 IKO embryos the DRG were analyzed by immunofluorescence for Islet1 expression, and the TG of the same embryos were used for microarray analysis. (A–C) Knock-down of Islet1 protein in cervical-level DRG at E14.5. Note that Islet1 expression is also missing from the motor area of the spinal cord (mot). Arrows in (B) show examples of a small number of neurons in which recombination has not occurred. (A) and (B) were processed on the same slide and photographed at the same exposure. In (C) LacZ expression is activated from a Rosa26-LacZ reporter allele by the induced Cre. (D,E) Islet2 expression in control and IKO E14.5 DRG. (F) Exon-specific Islet1 mRNA levels determined by qPCR in DRG of IKO embryo relative to control (1.0). Exon 4 is flanked by loxP sites. (G) Effect of late Islet1 excision on gene expression in the E14.5 TG. Genes are displayed in the order of fold decrease in E12.5 DRG (Table 2). Asterisks indicate significant increase or decrease (change p <0.002 or >0.998) in two independent comparisons. (H) Additional late Islet1 targets identified in E14.5 IKO TG.

Figure 7

Figure 7. Analysis of Islet1 induced knockout DRG at E18.5

Excision of the Islet1 homeodomain was induced by tamoxifen injection at E11.5 (Figure 6, Methods), and embryos were analyzed at E18.5. (A–C) Knockdown of Islet1 expression assessed by immunofluorescence. Islet1 expression is absent in the dorsal interneuron (dI) and motor neuron (mot) pools, and markedly diminished in the DRG. In (C), co-immunofluorscence for βgal expressed from the Rosa26-LacZ allele reveals a small population of Islet1+ neurons in which recombination has not taken place (red). In the spinal cord, βgal immunoreactivity identifies some surviving motor neurons. However, the dI interneurons are represented only by small, dense particles of βgal immunoreactivity, consistent with cellular debris (dashed line), suggesting that the dI population is dependent on Islet1 for survival. Matched sections were processed on the same slide and photographs were taken with the same exposure parameters. (D) Brn3a immunoreactivity was used to count neurons in lumbar DRG of Control and IKO embryos. Ten matched sections from control and IKO ganglia were counted, revealing no significant difference in neuronal number, and demonstrating that late expression of Islet1 is not required for DRG viability. Two-tailed T-test for difference between control and IKO, p = 0.93. (E–F) Runx1 immunoreactivity was near normal in IKO ganglia, in contrast to the marked decrease observed in CKO mice. (G–J) TrkA and Drg11 expression did not appear different in Control and IKO ganglia. (I–L) Expression of the nociceptive markers TrpV1 and TrpM8, evaluated by in situ hybridization, was diminished in IKO ganglia. TrpV1 cells/section: Control N=10, mean 5.9 ±3.1, range 2–12; IKO N=10, mean 1.2 ±1.2, range 0–3. T-test for difference between control and IKO, p =0.0003. TrpM8 cells/section: Control N=10, mean 16.8 ±5.3, range 11–23; IKO N=10, mean 5.3 ±2.5, range 2–10. T-test for difference between control and IKO p =3×10−7.

Comment in

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