1. Floor Plate Issues Diversity – Non-neuronal cells – Neurons Developmental origins Midbrain dopaminergic cells Comparison to insect midline cells

2. Spinal Cord Development

3. Notochord Induction of Floor Plate Formation in Mammals and Chicks Shh

4. Floor Plate: Shh Induction of Spinal Cord Development Shh Induction of Spinal Cord Development – Patterns ventral spinal cord – v3, MN, v2, v1, v0

5. Floor Plate: Control of Axon Crossing Control of axon crossing – Netrins – Shh

6. Zebrafish Floor Plate Description Early description (Kuwada 1990) – Consists of 3 rows of cells – Defined by cuboidal shape, and most extend no processes (non-neuronal) – Among earliest identifiable cells in spinal cord and appear before first growth cones – Crossing growth cones are between FP and basal lamina next to notochord

7. Zebrafish Floor Plate Description Two cell populations (Odenthal 2000) – Medial FP (MLP): shh +, netrin1 +, fkd4 +, fkd7 + – Lateral FP (LFP): foxa2 (fkh) +, fkd4 +, netrin-1 +

8. Zebrafish MFP Genetics MFP – cyclops (cyc; nodal-TGF-  ), one-eyed pinhead (oep; EGF-CFC), schmalspur (sur; foxh1) result in absence of MFP but not LFP LFP does not require MFP for development

9. Zebrafish LFP Genetics LFP – no-tail (ntl; fly brachyenteron), floating head (flh), momo (mom) have an absence of notochord flh or mom: MFP present with associated LFP; LFP absent when MFP absent In ntl: get expansion of MFP and LFP LFP can develop without notochord (as can MFP), but LFP only in presence of MFP

10. Shh Control of LFP syu (sonic you: shh) – Affects LFP but not MFP – Inject syu RNA  increase LFP but not MFP

11. Zebrafish Summary Early cells decide to be either notochord or FP MFP: notochord and shh-independent; requires nodal-related pathway (TGF-  ) LFP: dependent on shh from either Notochord or MFP Species differences: – Murine and chicken shh from notochord is required for induction of all FP cells – Murine Shh and netrin-1 are only in MFP and foxa2 (fly fkh) broadly expressed – Chicken Foxa2 in MFP and shh and netrin-1 in LFP

12. Multiple Floor Plate Cell types Zebrafish (Schäfer 2007) – foxa2: all MFP; subset LFP – tal2 bHLH gene (fly ~ HLH4C) in subset of foxa2 – LFP Insect comparison – Both insect midline and vertebrate FP consist of neurons and non-neuronal (glial) cell types

13. Tal2+ LFP cells are Postmitotic Label with pHH3 – Tal2 + cells negative – Foxa2 + tal2 – cells positive Tal2 + cells are postmitotic

14. Multiple Floor Plate Cell types Late stage embryonic FP – Sim1 + cells are LFP subset and postmitotic – Sim1 + cells express neuronal marker – Sim1 + cells are V3 interneurons (glutamatergic in other contexts) – Also observed GABAergic neurons

15. Summary Tal2 + cells are neuronal progenitor cells – Subset  sim1 + V3 interneurons – May also give rise to other interneurons Tal2 – foxa2 + cells are non-neuronal Insect comparison – Both insect ML and vertebrate have Sim + interneurons

16. Notch Signaling Mindbomb (Neur) mutant – Nkx2.2b (vnd), foxa2 (LFP non-neuronal cells), tal2 (LFP neurons) reduced or absent – Foxa2 in MFP (non-neuronal) normal Notch signaling required for LFP and p3 neurons wt mib

17. Shh Signaling In shh mutants, p3 LFP cells absent In dtr/+ and yot/+ foxa2 LFP was absent and tal2 and sim1 normal Thus, non-neuronal LFP cells require high Shh levels compared to p3 cells

18. Summary Early – foxa2 + cells proliferate – Tal2 + cells are postmitotic and differentiate into neurons Later – Subset of tal2 + cells become sim1 + V3 interneurons – Also GABAergic KA neurons (sim1 + ?) Notch signaling required for LFP and p3 cells Shh signaling: LFP non-neuronal and neuronal cells required different levels of Shh

19. Midbrain FP Cells Midbrain dopaminergic neurons – Motor control, reward, addiction, attention, cognition – Loss results in Parkinson disease Origins: mouse (Kittappa 2007) – TH + cells are in foxa2 + FP cells in midbrain

20. Midbrain FP Cells Shh is expressed in FP Shh-cre X R26R lacZ reporter: lineage marker TH + cells overlap with  -Gal

21. Development of Midbrain FP Cells Dorsal fate 5-HT1a fate

22. Midbrain FP Cells Progenitor cell development – Shh: induces FP – Otx2: required for generation of mesDA precursors – Lmx1a (CG32105): required for mesDA cell fate – Ngn2: proneural function Postmitotic development – Nurr1: controls subset of mesDA gene expression (TH) – Lmx1b (CG4328): survival; affects Pitx3 but not other genes (Nurr1, early TH) – Pitx3: lose TH expression, affects migration, survival – En: required for late maturation and survival

23. Foxa2 Kittappa 2007 Foxa2 –/– – No TH + cells Foxa2 +/– – Motor defects – Loss of DA neurons – Like PD

24. En En (Simon 2004) En is gradually expressed in TH + cells En –/– – Early TH + cells present but lack fibers – Late TH + cells absent

25. Summary Vertebrate FP:insect ML – Diversity of neuronal and non-neuronal cell types – MG = non-neuronal FP; both important signaling centers – Sim + cells present – DA + cells present – Genes?