amy huang research presentation - how can we identify novel motor neuron pool and column markers and...
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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!
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