1. Development PNS nervous system > neural crest derived > somatosensory neurons of the DRG > specialized for sensory modalities Somatosensory system > sense of body & environment on body > sensory modalities: touch, pain, temperature; proprioception > somatosensation integrated into spinal circuits > reflexes > somatosensory information sent to somatosensory cortex The sense of touch Importance of touch > neural crest derived > somatosensory neurons of the DRG > specialized for touch > Transcription factors driave differentiation of touch sensing neurons (cMaf; MafA) Summary so far …….

2. Species-specific neuronal circuits directed by neurotrophic factor control of transcriptional programmes > novel neural circuits arise during evolution to encode unique behaviors among different animal species.

3. How did somatosensory system evolve? What adaptations have been necessary in vertebrates? -Constant temperature (sea) > variable temperature (land) -Fins became limbs (circuits for coordinated movement) - Scales (fish), feathers (birds) & skin (mammals)

4. Chick Mouse > hopping gait = sautiller > feathers > alternating locomotion > hairs, a cold adaptation necessary for the prevention of heat loss. Expect species specific differences in spinal circuitry

5. Different innervation of skin in mammals and birds elaborate nerve endings in the epidermis no nerve endings in the epidermis

6. Diversité des neurones sensoriels périphériques des ganglions rachidiens Marmigère and Ernfors Nature Reviews Neuroscience 8, 114–127 (February 2007) TrkB et/ou c-Ret TrkC TrkA ou c-Ret Moelle épinière Ganglion Rachidien Dorsaux (DRG) Neurones prorioceptifs Neurones mécanoceptifs Neurones nociceptifs & thermosensitifs

7. Thermo- nociception Mechano- ception Proprio- ception

8. Spinal cord Dorsal root ganglion (DRG) Proprioceptors TrkC/NT-3 Nociceptors Thermoceptors TrkA/NGF Ret/GDNF Mechanoreceptors TrkB/BDNF Somatosensory neurons are highly diverse

9. Neurotrophins and sensory neuron development > survival > maturation > axonal projections

10. Intrinsic versus extrinsic signals for neuronal differentiation

11. Da Silva & Wang 2010 Curr. Op. Neurobiol Concept: Intrinsic & extrinsic signals co-ordinate sensory neuron – spinal neuron interactions

12. Figure 2. Peripheral signals control the formation of dorsal root ganglion sensory and motor neuron projections. (a,b) Subpopulations of brachial motor neurons extend their axons towards their target muscles. En route, they encounter glial-cell-line-derived ne... Simon Hippenmeyer, Ina Kramer, Silvia Arber Control of neuronal phenotype: what targets tell the cell bodies Intrinsic versus extrinsic signals for neuronal differentiation

14. Concept: A species-specific signal controls nociceptive circuit formation HoxD1 NGF Spinal cord neuron Nociceptive neuron TrkA DRGSpinal cord Skin

15. Summary Hypothesis Species are endowed with unique sensory capabilities encoded by divergent neural circuits. One potential explanation for how divergent circuits have evolved is that conserved extrinsic signals are differentially interpreted by developing neurons of different species to yield unique patterns of axonal connections. Although NGF controls survival, maturation and axonal projections of nociceptors of different vertebrates, whether the NGF signal is differentially transduced in different species to yield unique features of nociceptor circuits is unclear. Results We identified a species-specific signaling module induced by NGF and mediated by a rapidly evolving Hox transcription factor, Hoxd1. Mice lacking Hoxd1 display altered nociceptor circuitry which resembles that normally found in chicks. Conversely, ectopic expression of Hoxd1 in developing chick nociceptors promotes a pattern of axonal projections reminiscent of the mouse. Conclusion We propose that conserved growth factors control divergent neuronal transcriptional events which mediate interspecies differences in neural circuits and the behaviors they control. The form of a scientific paper

16. NGF and sensory neuron development Nociceptors - pain - temperature - touch - itch > survival > maturation > axonal projections

17. Hox genes in development Hox genes pattern the rostro-caudal axis

19. HoxD family – role in limb patterning (medio-lateral axis)

21. 1. Search for transcriptional targets of NGF different between birds and mammals i. Genes enriched in nociceptors ii. NGF-regulated genes expressed in nociceptors in vivo iii. mouse DRG explants grown in the presence or absence of NGF for identification of NGF-dependent genes expressed in nociceptors in vitro. > NGF dependent genes in mouse nociceptors Mouse DRG culture Chick DRG culture + NGF > differentially regulated genes detected by Q-PCR HoxD1

22. Results 1. A screen for genes controlled by NGF signalling in mammalian nociceptors HoxD1 is an NGF regulated in mouse, but not in chick

24. Fig. 1 HoxD1 expressed in mouse nociceptors (TrkA+), but not in chick

25. > HoxD1/TrkA co-expression is specific to mouse 2. Developmental expression of Hoxd1 in different vertebrate species

27. WT HoxD1 -/- Nociceptive innervation of the skin of Hoxd1−/− mice resembles that of non-mammalian vertebrates e.g. birds (in B loss of circular endings around hair follicles) 2. Developmental expression of Hoxd1 in different vertebrate species

28. Abnormal expression of Mrgbp4 in peptidergic neurons in HoxD1-/- mice

29. 3. Hoxd1 instructs nociceptor central axonal projections within the mammalian spinal cord Mirroring species-specific differences in innervation of the skin, the patterns of nociceptive axonal projections within the spinal cords of mammals and birds are also distinct > ectopic innervation of deep layers of the spinal cord

30. Different patterns of nociceptor innervation of the spinal cord in mouse and chick

31. 4. Hoxd1 instructs nociceptor central axonal projections within the mammalian spinal cord - suggests that Hoxd1 mediates NGF-dependent suppression of nociceptor projections into deep layers of the spinal cord.

32. In Mouse Loss of NGF or Loss of HoxD1 > “chick-type” skin & spinal cord innervation What is effect of expressing HoxD1 in chick DRG neurons? The gain-of-function experiment

33. Electroporation of plasmid DNA into chick embryo neural tube Chick as a model for study of spinal cord circuitry Negatively charged DNA moves towards the anode

34. 5. Ectopic Hoxd1 expression in chick nociceptors induces mammal-like traits > HoxD1 expression in chick suppressed deep layer nociceptor innervation in chick

35. HoxD1 mutation changes nociceptor circuitry in the mouse spinal cord Physiological consequences?

36. 6. Hoxd1−/− mice have deficits in cold sensitivity

37. Conclusion > Hoxd1 instructs development of mammal-specific features of nociceptive neural circuitry. > behavioral sensitivity to extreme cold is markedly compromised in Hoxd1 mutant mice > suggests HoxD1 was co-opted by nociceptors in mammals for cold sensation

38. The end