Prediction of active nodes in the transcriptional network of neural tube patterning

Abstract

A transcriptional network governs patterning in the developing spinal cord. As the developmental program runs, the levels of sequence-specific DNA-binding transcription factors (SSTFs) in each progenitor cell type change to ultimately define a set of postmitotic populations with combinatorial codes of expressed SSTFs. A network description of the neural tube (NT) transcriptional patterning process will require definition of nodes (SSTFs and target enhancers) and edges (interactions between nodes). There are 1,600 SSTF nodes in a given mammalian genome. To limit the complexity of a network description, it will be useful to discriminate between active and passive SSTF nodes. We define active SSTF nodes as those that are differentially expressed within the system. Our system, the developing NT, was partitioned into two pools of genetically defined populations by using flow sorting. Microarray comparisons across the partition led to an estimate of 500 -700 active SSTF nodes in the transcriptional network of the developing NT. These included most of the 66 known SSTFs assembled from review articles and recent reports on NT patterning. Empirical cutoffs based on the performance of knowns were used to identify 188 further active SSTFs nodes that performed similarly. The general utility and limitations of the population-partitioning paradigm are discussed.Lbx1 ͉ microarray ͉ mouse ͉ spinal cord S pinal cord development begins when the neural tube (NT) forms from a sheet of proliferative neuroepithelium. The proliferative ventricular zone around the lumen sheds postmitotic cells into a mantle layer from embryonic day (E)9.5 to E13.0 of mouse development. Many sequence-specific DNA-binding transcription factors (SSTFs) that mark discrete neuronal subpopulations during ontogeny have been identified (1-10). SSTFs also mark subdivisions in the motor neuron (MN) pool (11-15) and mark different types of ventral interneurons (16). Gene knockout experiments show that SSTFs specify neuronal types (17-21). Mutating them often leads to respecification of populations. The Lbx1 homeobox gene contributes to the specification of at least four spinal cell types. Loss of Lbx1 function leads to an organized respecification of transcription factor codes and projection patterns in Lbx1-marked populations (22, 23). It therefore is thought that a network of SSTFs can be used to describe patterning and specification in the NT.The neuronal diversity generated by patterning mechanisms in the mouse NT between E9.0 to E13.0 is not greatly influenced by classic synaptic function. Voltage-dependent Na ϩ conductance first appears at E12 (24), periodic MN bursting begins at E12.5, and a full locomotor pattern generator is not observed until E16.5 (25). Electrical connectivity during the patterning phase of development appears to be limited to gap junctions (26). In addition, the primary afferent neurons begin to enter the dorsal horn only at the end of this phase, suggesting that there is little sensory contact with the external environment. The absen...

How to Build Transcriptional Network Models of Mammalian Pattern Formation

“…Thirty of the 141 SSTF target genes have already been implicated in neural tube development and 85 were predicted active nodes [19]. Only 26 targets were neither known nor predicted nodes.…”

Population-Specific Regulation of Chmp2b by Lbx1 during Onset of Synaptogenesis in Lateral Association Interneurons

“…Neural tubes were dissected from heterozygous (Lbx1 GFP/+ ) and mutant (Lbx1 GFP/GFP ) embryos at E12.5. Dissected neural tubes were dissociated and sorted into GFP + (green) and GFP − (white) cell types as previously described [25]. RNA from these cells was used to probe Affymetrix U430 2.0 microarrays.…”

“…The array data showed that all 30 of the other ESCRT RNAs were expressed at identical levels in green and white cells (data not shown), which according to the population partitioning model [25], indicated that they are uniformly expressed in the neural tube. The arrays also showed that none of the other ESCRT genes were Lbx1 -dependent.…”

“…Immunohistochemistry, flow sorting, microarray sample preparation, microarray processing, microarray analysis, and qPCR confirmation procedures, as well another subset of the data of the indicated arrays have been described previously [25], [30]. For the present report we developed a new in situ hybridization procedure for mouse embryos that uses RNA probes prepared with biotinylated–UTP.…”

“…The postmitotic daughter neurons that emerge laterally from each spatial domain express new, distinct combinations of SSTFs. Population partitioning analysis has been used to estimate that at 30–45% of the SSTFs in the mammalian genome have spatially constrained expression patterns in the neural tube [25]. A combinational code of SSTF expression is used to define populations of neurons along both the dorsal–ventral and anterior-posterior axes in both the ventricular and mantle zones [26], [27], [28].…”