Periphery-derived TGF-β signaling orchestrates the formation of topographical maps in the brain

The mouse trigeminal system is an extremely well-characterized and accessible model for neuronal development, sensorimotor integration, and active sensation. Rodent whiskers are tactile sensors that can detect subtle differences in amplitude, velocity, orientation, and duration of a tactile stimulus. Several subtypes of mechanosensory neurons innervate each whisker follicle and the neurons representing a single follicle project together to form a barrelette, a synaptic structure in the brainstem representing a single whisker. Barrelettes are spatially arranged in a way that exactly parallels the spatial organization of the whiskers themselves.

Fig. 1. Trigeminal sensory neurons innervating neighboring whiskers are intermingled and scattered in the TG. (A) Schematic diagram of the topo- graphic arrangement of large whiskers in the mouse face. Whiskers are or- ganized in five rows (A–E) along the D-V axis and in up to seven arcs (1–7) along the A-P axis. (B) D2 and D3 whiskers were injected with Dextran- fluorophore tagged with either Alexa568 or Alexa488 at P0. Injections resulted in labeling of cell bodies and axon projections of trigeminal sensory neurons. (C) Horizontal and sagittal sections of TG 3 d after injection of mice with dual-labeled D2 and D3 whiskers reveal that cell bodies of neurons innervating adjacent whiskers are intermingled in the ganglion. (D) Coronal sections of SpC, SpI, and PrV nuclei at P3 reveal clear segregation of central axon projections of D2- and D3-innervating neurons into two distinct barrelettes. Note that the whisker map in the SpC is represented with an opposite orientation compared with the maps in the SpI and PrV. BS, brainstem; CT, central tract; DexA488, Dextran-Alexa488; DexA568, Dextran- Alexa568. (Scale bars: C, 100 μm; D, 50 μm.)

Using a dual-color iontophoretic labeling strategy, Wang determined that while the central projections of the mechanosensory afferents are clearly segregated into barrelettes in the brainstem, their cell bodies are dispersed and intermingled in the trigeminal ganglion (Fig 1). Therefore, the precise topographical representation of the whisker map in the brainstem is not a result of simple presegregation of the axonal projections exiting the sensory organ within the ganglion.

Dr. Wang’s lab has previously demonstrated that bone morphogenetic protein 4 (BMP4), one member of the transforming growth factor beta (TGF-β) superfamily of structural proteins, is an early developmental retrograde signal that affects trigeminal sensory neuron identity through the induction of transcriptional changes. These neuronal identities affect the formation of topographical facial maps. This hinted at the potential importance of the expression pattern of TGF-β proteins during the period of follicle development and innervation.

Using in situ hybridization, several TGF-β ligands (e.g. Activin A) that are exclusively expressed in the periphery were identified. Furthermore, this expression pattern is spatiotemporally dynamic and the time period exactly overlaps with the formation of whisker maps in the brainstem. Because the binding of TGF-β ligands with their receptors induces the phosphorylation of Smad2, levels of phospho-Smad2 (pSmad2) were analyzed as a biomarker for TGF-β signaling in developing trigeminal sensory neurons. The pSmad2 signal was detected in the peripheral tract of the trigeminal ganglion during the period of whisker development in late embryos. To further test the role of TGF-β signaling in barrelette map formation the gene encoding Smad4, a downstream component of the TGF-β signaling cascade, was selectively deleted in sensory neurons. The Smad4flox/flox mice were crossed with a sensory-specific Cre line to ensure spatial specificity of the deletion while preserving normal follicular development (Fig 3A). Cytochrome oxidase (CO)-staining of brainstem sections revealed small and irregularly-shaped barrelettes in the trigeminal principalis (PrV) and spinal interpolaris (SpI) brainstem nuclei and the complete lack of barrelettes in the spinal nucleus caudalis (SpC) of Smad4 knockouts as compared to controls (Fig 3C).

Fig. 3.  Smad4-cKO sensory neurons form defective barre- lettes. (A) Schematic of the alleles used to generate [Advil- lincre/+; Smad4flox/flox] mice: AdvillinCre, Smad4flox, and ROSA26Sortm1(CAG-ALPP)Fawa (RosaPLAP). (B) In situ hybridization of Smad4 in Smad4-cKO mice at E15.5 confirms the deletion of Smad4 in trigeminal sensory neurons. Magnified view (Right) of the regions highlighted (Left). (C) CO staining of control and Smad4-cKO SpI nucleus at P1, P5, and P7. Note that at P1, barrelettes are distinguishable in control SpI (arrows) but imperceptible in Smad4-cKO SpI. At later stages, barrelette-like structures emerge in Smad4-cKO SpI, but de- fined barrelettes are never observed. (D) TenascinC immu- nostaining (green) of a P7 SpC nucleus. In control mice, barrelette boundaries are revealed by TenascinC (arrow- heads), whereas in Smad4-cKO mice, this pattern is lost. (E) vGlut1 immunolabeling (red) of PrV, SpI, and SpC of P7 control and Smad4-cKO mice. vGlut1 staining reveals the emergence of barrelette-like structures in SpI and PrV nuclei and no bar- relettes in the SpC nucleus of Smad4-cKO mice. (Scale bars: 100 μm; B, enlarged views, 25 μm.)

Immunofluorescence revealed that Smad4-deficient neurons exhibit normal whisker innervation. The viability and differentiation state of these neurons was shown to be normal, and no axonal outgrowth differences between Smad4-deficient neurons and control neurons were observed. The abnormal barrelette phenotype observed is, instead, attributable to the misprojection and deficient segregation of trigeminal neuron central projections into appropriate barrelettes. Using the same dual-color iontophoretic labeling strategy as before, central projections from the populations of neurons innervating adjacent neurons were traced. Smad4-cKO axons project to a larger and more diffuse area when compared with the projections of control neurons, especially in the SpI nucleus. There is deficient segregation of these projections into barrelettes in all brainstem nuclei analyzed of Smad4-cKO, compared to controls. Additionally, morphological analysis revealed that Smad4-cKO neurons have simpler terminal arbors and form fewer synapses than control neurons, a phenotype that is likely a consequence of axon misprojection.

In short, these findings demonstrate that TGF-β signaling in trigeminal sensory neurons is necessary for the proper formation of the barrelette map. The plasticity of this sort of periphery-derived retrograde signaling mechanism makes it a likely candidate for the regulation of innervation and mapping of complex sensory organs by the somatosensory and motor systems of different mammalian species.

Fan Wang

Please join us for the seventh installment of the 2012-2013 Neuroscience Seminar series at 4 pm on Tuesday, November 13h in the CNCB Large Conference Room, as Fan Wang shares her insights into the sensory and motor circuitry of active touch.

Kelsey Mason is a first year in the neurosciences Ph.D. program. She is completing her first rotation with Dr. Ursula Bellugi and Dr. Eric Halgren.

da Silva S., Hasegawa H., Scott A., Zhou X., Wagner A.K., Han B.X. & Wang F. (2011). Proper formation of whisker barrelettes requires periphery-derived Smad4-dependent TGF-  signaling, Proceedings of the National Academy of Sciences, 108 (8) 3395-3400. DOI: