How can we identify novel motor neuron pool and column markers and specific spinal neuron subsets?

Amy Huang, Kevin Kanning, and Christopher Henderson

Goals

To discover cell-surface antigens that could be used for Ab purification of MNs

To discover novel phrenic and hypaxial gene markers

To discover novel MN markers that are transcription factors (TFs)

To identify novel cranial (hindbrain) and spinal cord pool markers

To improve the molecular roadmap of the embryonic spinal cord

To consolidate expression data from online mouse databases into a comprehensive database

Create reference atlas Create outline of Excel database with ranking scale Small-scale pilot screen with initial search

Initial search – Genepaint search on brain and spinal cord

2nd search – Transcription factors 3rd search – Eurexpress search on brain and spinal cord 4th search – Cluster of differentiation (CDs) 5th search – Neuropeptides, microRNA, and finally, Genome

Procedure

Rationale: Obtain usable data quickly and efficiently

Reference Atlas For identification of MN pools and columns

All MN – Chat, Isl2, Ngfr, Isl1 Pools – Runx1, BMP1, Cdh9 (for phr), Pappa (for phr), Nts

Pappa – E14.5

Er81 – E14.5

Excel databaseScale composed of Specificity (1-5) and

Signal (1-5). The Sum is the total of both.

Specificity: how “specific” is the

expression. Diffuse?

Localized?

Signal: Strength of expression.

Strong? Moderate?

Weak?

Initial Screen - Genepaint

Second Search – TFs

Useful since TFs go under various

names

Science. 2004 Dec 24;306(5705):2255-7.

Third Search - Eurexpress

Other Searches

Cluster of Differentiation (CDs) Neuropeptides microRNA GENOME

Phrenic candidates

Atp8a1 ATPase, aminophospholipid transporter (APLT), class I, type 8A, member 1

Dhcr24 24-dehydrocholesterol reductase

Lgi3 leucine-rich repeat LGI family, member 3

Phrenic candidates

LOC434147 Ndrg4 N-myc downstream regulated gene 4

Thy1 thymus cell antigen 1, theta

All MN candidates

JunJun oncogene

Rtn4 1110054N06Rik 0610012G03Rik 2310003P10Rik 3110006E14Rik Ache Acly Acot7 Adcy2 Aldoa Ankrd6 Ap2m1 Arhgdig Armcx1 Armcx3 Asns Atp2b2 Atp5j2 Atp6v1f AW539457 bcl11b 2310042M24Rik Calcb Capns1 Cd81 Cdipt Ceacam9 Chat Chodl Cib2 cops5 Cops5 Cplx1 Cycs Dhcr24 Efnb3 Emb Evi1 Fgf1 Ghitm Gpi1 9630031F12Rik Homer2 Hsp90aa1 Isl1 Jak1 Jun Laptm4b Lbxcor1 Nef3 Lgi3 Lifr Lims1 Lix1 Lrp11 Lrsam1 Lynx1 Mdh1 Mdh2 Melk Mfap3l Mmp15 Mnx1 Mtap Mtpn Ndel1 Ndrg4 Negr1 Ngfr Nrg1 Pcbp3 Pde10a Pfkp Plxna3 Ppp2r2b Prdx1 Prkar1b Ptdss1 Rabac1 Rasgef1c Reps1 Resp18 Rgs11 Rtn2 S100a10 S100b sap18 Scg2 Scg3 Scrn1 Selm Sema3c Serpinb6a Sgcb Sh3bgrl3 Slc10a4 Slc18a3 Slc25a1 Slc35e4 D15Mgi27 Slit2 stat3 Syt11 Tpd52 Trim2 Tspan17 Ttl twist1 Ube4b Vdac2 wig1 zfr zhfx1a zim1

~ 113+ Genes

M7 candidates 34 total genes

A330102H22Rik (Prune2)E14.5

Atf3activating transcription factor 3

Vipvasoactive intestinal polypeptide

M7 candidates

Pyypeptide YY

Serpina3kserine (or cysteine) peptidase inhibitor, clade A, member 3K

Neo1Neogenin

M7 candidates

Etv1 (Er81)ets variant gene 1

Esrrb estrogen related receptor, beta

Nrsn1 neurensin 1

Cell-adhesion molecule candidates

Tspan17 tetraspanin 17

Ceacam9 carcinoembryonic antigen-related cell adhesion molecule 9

HMC candidates

Gss: glutathione synthetase

HMC candidates

Slc17a4 solute carrier family 17 (sodium phosphate), member 4

Mmu-miR-218

SummaryTotal genes screened – ~ 3,597 Total genes annotated – 1,997

168 failed probes107 no Genepaint data43 NCBI search null22 little to no spinal expression292 no expression77 ubiquitous

113+ all MN246 M7105 M7 pools4 floorplate4 HMC50 interneuron

15 lumbar pools1 M36 M123 M53 phrenic3 M9/M10750 no MNs

113

246

105 44

50

1

3

15

3

750

All MNM7M7 poolfloorplateHMCInterneuronM3M9/M10lumbar poolphrenicno MN

Goals – Revisited

To discover cell-surface antigens that could be used for Ab purification of MNs5+ specific genes

To discover novel phrenic and hypaxial gene markers7+ specific genes

To discover novel MN markers that are transcription factors (TFs)38+ genes

To identify novel cranial (hindbrain) and spinal cord pool markers136+ pools

To improve the molecular roadmap of the embryonic spinal cord

To consolidate expression data from online mouse databases into a comprehensive database

Future Goals and Improvements

Improve scale to be more objective Perform in-situ on interesting candidates Screen microRNA when Eurexpress completes its

database Complete the mouse genome screen

Many Thanks!