Patterning the Brain: Local Signaling Centers and Notochord Role

KING'S College LONDON

Faculty of Life Sciences & Medicine

11/2024 Esther Bell esther.bell@kcl.ac.uk Department of Anatomy

Patterning the Brain 2Local Signalling Centres

• gradients (morphogens)
• local signalling centres "organisers" isthmus and anterior neural boundary (ANB)
• segmentation, boundaries
• signalling centres in the forming brain

Regions in the Anterior Brain

Regions in the anterior brain are defined by domains of transcription factor expression in the neural plate anterior anr zli forebrain te Six3 W/ht anterior pcp Irx3 midbrain ot Otx2 isO ce hindbrain Fgf8 posterior Shh ---- Gbx2 posterior

Transcription Factor Expression in the Neural Plate

Regions in the anterior brain are defined by domains of transcription factor expression in the neural plate anterior Otx2 midbrain isO Gbx2 hindbrain posterior

Cross Repression of Transcription Factors

Cross repression of transcription factors create a sharp border

Boundary Areas and the Isthmic Organiser

Boundary areas becomes Secondary Signaling Centre: the case of the Isthmic Organiser (midbrain/hindbrain boundary=MHB) anterior Otx2 Gbx2 posterior A B Otx2 HH4 Gbx2 HH4 G J ch4 Otx2 HH10 Gbx2 HH10

Isthmic Organiser Position and Gene Expression

The interphase between Otx2 and Gbx2 defines the position of the Isthmic Organiser (aka MHB) and the expression of Fgf8 and Wnt1 K Fgf8 HH10 I Mes Met Otx2 E9.5 T Wnt1 r2-4 Gbx2 Fgf8 mes met Gbx2 Otx2 Wnt1 Fgf8 A mb B di hb mx ma tb SC ht E9.0 2 St.15 (24 somites) Wnt1 FGF8

Functional Role of the Isthmus

What is the functional role of the isthmus?

Isthmus Requirement for Cerebellum and Midbrain Patterning

The isthmus is required to pattern both the cerebellum and midbrain forebrain midbrain hindbrain can test this experimentally ... Stage 10 Adult Ectopic cerebellum Ectopic midbrain Quail donor Chick host

AP Rotation of the Isthmus

AP rotation of the isthmus FB MB HB Mes2 Mes 1 Mes Cb2 Di Cb1 Tel Cb HB Rubenstein and Puelles, 1994

Genetic Test of Otx/Gbx Role by Loss of Function

Genetically test role of Otx/Gbx by loss of function Otx2-/- no brain anterior to hindbrain Mes Met Otx2 E9.5 T Wnt1 r2-4 Gbx2 Fgf8 mes met Gbx2 Otx2 Wnt1 Fgf8 Gbx2-/- no hindbrain What about Fqf8 and Wnt1?

FGF8 Expression in Isthmic Organiser

Expression of FGF8 in isthmic organiser is required for formation of midbrain and cerebellum Wild type Mes Met Otx2 E9.5 T Wnt1 r2-4 Gbx2 Fgf8 mes met Gbx2 Otx2 Wnt1 Fgf8 di: Diencephalon tc: Midbrain cb: Cerebellum Fgf8 mutant di mb cb di * Shanmugalingam et al, 2000

Wnt1 Expression in Isthmic Organiser

Expression of Wnt1 in isthmic organiser is required for formation of midbrain Wild-type Wnt1 -/- Mes Met Otx2 E9.5 T Wnt1 r2-4 Gbx2 Fgf8 mes met Gbx2 Otx2 6 my my Wnt1 Fgf8 C ch mb ch * di cb di cp D cb: Cerebellum di: diencephalon mb: midbrain my: myencephalon (hindbrain) Crossley and Martin, 1995

Forebrain Subdivision by ANR

The forebrain is subdivided into areas by acquisition of positional info. given by local signalling centre: ANR fgf wnt ANB: Anterior Neural Border

ANR Requirement for Forebrain Patterning

ANR is required for forebrain patterning dlx - ventral forebrain emx - dorsal forebrain a b - C d t d mb - Removal of ANR results in no telencephalon (Emx1 expression) d a A Row 1 Row 1 row 1 t d b row 1 hy ap Donor Host - f t d mb - g h experimental: row 1 - Houart et al, 1998 What is the signal(s) secreted from the ANR? control: row 3 C

ANB Signal: Wnt Antagonist

One key ANB signal is a Wnt antagonist Frizzled SFRPs (frzbs) NH2 cysteine-rich signal hydrophilic 200 COOH netrin-like transmembrane cytoplasmic COOH Wnt sfrp Wnt LRP6 LRP6 P P P P Axin P P Dishevelled CK1y P Axin P P APC P GSK-3 ß-catenin degradation Cytoplasm HDAC CTBP TCF Nucleus Wnt absent B-catenin degradation inhibited APC P GSK-3 ß-catenin degradation Cytoplasm HDAC CTBP TCF XD Nucleus Wnt present CK1y P Axin P P Dishevelled GSK-3 CK1y Secreted Frizzled Related Protein N112 cysteine-rich signal Wnt inhibited Gro TCF free B-catenin accumulates

Wnt Inhibitor Expressed in Row 1

tlc - wnt inhibitor expressed in row 1 B C 65% tic 95% tle tlc H 10pg J 50pg L 100pg cmx 1 emx 1 emx 1 Houart et al, 2002 tlc rescues ablation of row 1

Brain Patterning Summary

Patterning the Brain

• gradients (morphogens)
• local signalling centres "organisers" forebrain (ANR), midbrain (MHB)
• segmentation, boundaries
• signalling centres in the forming brain

Formation of Brain Compartments

Formation of brain compartments prosencephalon ANR PrZLI mesencephalon prechordal plate midbrain- hindbrain boundary (MHB) ,r1 r2 3 rhombencephalon r4 r5 r6 rotochord r7 Midbrain tectum MHB thalamus ZLI prethalamus cerebellum telencephalon Hindbrain medulla ventral midline motor nuclei notochord

Summary of Signaling Centres in the Neural Plate

Summary of signaling centres in the neural plate: isthmus and ANR tlc prosencephalon fgf ANR PrZLI otx2 mesencephalon otx2 prechordal plate gbx2 midbrain- hindbrain boundary (MHB) wnt1 fgf gbx2 r3 rhombencephalon r4 r5 r6 - notochord r7

Brain Compartments: Hindbrain Segmentation

Brain compartments: hindbrain segmentation in rhombomeres Cranial region of chick embryo (stage HH18, 3 days) rhombomeres of hindbrain midbrain r1 optic vesicle r2 r3 V r4 r5 VII r6 r7 forebrain r8 s2 b1 somites 1 b3 b4 branchial arches b2

Cell Movement Between Compartments

Once boundaries are formed, cells can not move between compartments Single neuroepithelial cells labeled at early and late stages and the labeled clones mapped at 2 days Early-stage labeling Pattern of clones Late-stage labeling r1 C C r7 Lumsden, 1991 Rhombomeres are cell-tight compartments within which neural precursors acquire their positional identity

Rhombomere Definition by Homeoprotein (Hox) Expression

Rhomobomeres are defined by Homeoprotein (Hox) expression anterior posterior fbed, zen lab pb Dfd Scr (ftz) Antp Ubx abd-A Abd-B Drosophila Hox complex Hox1 Hox2 Hox3 Hox4 Hox5 Hox6 (central) Hox7 (posterior) ancestral Hox complex + A1 A2 A3 A4 A5 A6 A7 A9 A10 A11 A13 B1 B2 B3 B4 85 B6 B7 B8 B9 B13 HOXB C4 C5 C6 C8 C9 C10 C11 C12 A13 HOXC D1 D3 D4 D8 D9 D10 D11 D12 D13 HOXD hindbrain and spinal cord mesoderm anterior posterior 1gX r8 omXII gxl r4 HOXBf HOXAT gVII HOXB3 HOXA3 r5 mVI OV rô mix b3 HOXB4 HOXA4 glx r7 HOXC4 mx Posterior Motor nerves and neural crest Sensory ganglia r1 FF HOXA2 r2 gv 1 mv b1 HOXB2 r3 mammalian Hox complex HOXA gVill b2 Anterior Nature Reviews | Neuroscience

Hox Gene Expression and Rhombomere Boundaries

Expression hox genes respect rhombomere boundaries r3 Hoxb-3 to r3/r4 boundary r4 r5 Hoxa-3 to r4/r5 boundary VI Hoxd-4 to r6/r7 boundary XII hoxa3 hoxb3 VI r6 r7 XII hoxd4 r4 r5 VI XII

Hox Genes and Rhombomere Identity

Hox genes give rhombomeres their identity: manipulation of the Hox code A BEN B GATA2 C Dil B r2 r3 r4 r5 r4 - F G H r2 RCAS r4 r4 r4 Overexpression of Hoxb1 in r2 transforms r2 to an r4 (facial) identity control r2 r4 r4 r6

Hoxb1 Requirement for r4 Identity

Hoxb1 is required for r4 identity D Dil/DIO r2 r3 bm V fp r2 mV - r3 r4 r4 mVII r5 r5 2 bm VII Normal Ectopic Hoxb 1 Hoxb1 +/- Hoxb1 -/- r4 MOUSE CHICK r4 r3 cvan I r2 r3 Studer et al 1996; Bell et al, 1999

Signaling Centres in the Forming Brain

Signaling Centres in the forming brain

• gradients (morphogens)
• local signalling centres "organisers" forebrain (ANR), midbrain (MHB)
• segmentation, boundaries
• signalling centres in the forming brain

Dorso-ventral Organisation of the CNS

Dorso-ventral organisation of the CNS: insight from the spinal cord Midbrain tectum MHB thalamus ZLI prethalamus cerebellum Forebrain telencephalon Hindbrain medulla ventral midline motor nuclei notochord

Notochord as Ventral Spinal Cord Organiser

The notochord is an organiser of the ventral spinal cord (a) (b) (c) (d) (e) O 00 000 0 00 00 @ 1995 Current Biology

Notochord: Induction of Floor Plate and Motor Neurons

Notochord: induces floor plate and motor neurons Normal differentation Removal of the notochord leads to a failure of differentation. Grafting an additional notochord induces addtional floorplate and motor-neuron differentiation. Motor neurons Floorplate Notochord Q

Notochord and Floor Plate Secretion of Sonic Hedgehog (Shh)

The notochord and floor plate secrete Sonic hedgehog (Shh) a Shh RNA b Shh protein

Signaling Centres: Roof Plate and Floor Plate

2 signaling centres: roof plate and floor plate Dorsal neural tube notochord Ventral Shh BMPS BMP causes a cascade of gene expression: TGFB family members Shh diffuses dorsally as a gradient

Neuronal Identity Determination by Molecular Concentration

Neuronal identity determined by concentration of molecules which in turn induce different transcription factors Dorsal A D Wnt BMP Dorsal Bmp Wnt d12 Roof plate dl3 Neural tube dl4 Somite RA dl5 d16 vO Floor plate v1 Notochord Shh V Ventral V Shh v3 FP Ventral v2 MN RP dl1

Expression of Class I and II Transcription Factors

Expression of Class I and II transcription factors A Pax7 Dbx2 Irx3 Pax6 Class I Class I: ventral boundaries Nkx6.1 Nkx2.2 Pax7 Dbx1 Dbx2 Irx3 Pax6 d Class II: dorsal boundaries Class II po p1 p2 pMN p3 Nkx6.1 Nkx2.2 Briscoe et al. 2000. Cell. 101. 435-445.

Shh Signalling and Protein Expression

Dorsal Pax7 Dbx1 Dbx2 Irx3 Pax6 Shh signalling po VO p1 V1 p2 V2 pMN MN Gli Shh p3 V3 Ventral Nkx6.1 Nkx2.2

• Shh signaling represses class I and activates class II protein expression
• Cross-repressive interactions between class I and class II proteins that abut a common progenitor domain boundary refine and maintain progenitor domains
• The profile of expression of class I and class II proteins within an individual progenitor domain controls neuronal fate Jessell. Nat Rev Genet. 2000. 1: 20-29 5 1 - -

Conclusions on CNS Patterning

Conclusions

• The major patterning decisions in the central nervous system are taken very early in development, prior to or very early after neural tube closure
• The subsequent refinement in cell identity is made by local changes in positional information thanks to the action of local signalling centres
• Formation of segments (and boundaries) is a very successful mechanism by which patterning decisions are "locked" and signalling can be restricted to defined directions

Reading List

Reading list Principles of Development 5th Ed Chapter 4 - Vertebrate development II: Xenopus and zebrafish Chapter 12 - Development of the nervous system Kiecker and Lumsden Annu Rev Neurosci. 2012; 35:347-67. The role of organizers in patterning the nervous system. Read