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Sequence specific transcription factors (SSTFs) combinatorially define cell types during development by forming recursively linked network kernels. Pitx2 expression begins during gastrulation, together with Hox genes, and becomes localized to the abdominal lateral plate mesoderm (LPM) before the onset of myogenesis in somites. The somatopleure of Pitx2 null embryos begins to grow abnormally outward before muscle regulatory factors (MRFs) or Pitx2 begin expression in the dermomyotome/myotome. Abdominal somites become deformed and stunted as they elongate into the mutant body wall, but maintain normal MRF expression domains. Subsequent loss of abdominal muscles is therefore not due to defects in specification, determination, or commitment of the myogenic lineage. Microarray analysis was used to identify SSTF families whose expression levels change in E10.5 interlimb body wall biopsies. All Hox9-11 paralogs had lower RNA levels in mutants, whereas genes expressed selectively in the hypaxial dermomyotome/myotome and sclerotome had higher RNA levels in mutants. In situ hybridization analyses indicate that Hox gene expression was reduced in parts of the LPM and intermediate mesoderm of mutants. Chromatin occupancy studies conducted on E10.5 interlimb body wall biopsies showed that Pitx2 protein occupied chromatin sites containing conserved bicoid core motifs in the vicinity of Hox 9-11 and MRF genes. Taken together, the data indicate that Pitx2 protein in LPM cells acts, presumably in combination with other SSTFs, to repress gene expression, that are normally expressed in physically adjoining cell types. Pitx2 thereby prevents cells in the interlimb LPM from adopting the stable network kernels that define sclerotomal, dermomyotomal, or myotomal mesenchymal cell types. This mechanism may be viewed either as lineage restriction or specification.

Background: Genetic regulatory networks of sequence specific transcription factors underlie pattern formation in multicellular organisms. Deciphering and representing the mammalian networks is a central problem in development, neurobiology, and regenerative medicine. Transcriptional networks specify intermingled embryonic cell populations during pattern formation in the vertebrate neural tube. Each embryonic population gives rise to a distinct type of adult neuron. The homeodomain transcription factor Lbx1 is expressed in five such populations and loss of Lbx1 leads to distinct respecifications in each of the five populations., Methodology/principal Findings: We have purified normal and respecified pools of these five populations from embryos bearing one or two copies of the null Lbx1(GFP) allele, respectively. Microarrays were used to show that expression levels of 8% of all transcription factor genes were altered in the respecified pool. These transcription factor genes constitute 20-30% of the active nodes of the transcriptional network that governs neural tube patterning. Half of the 141 regulated nodes were located in the top 150 clusters of ultraconserved non-coding regions. Generally, Lbx1 repressed genes that have expression patterns outside of the Lbx1-expressing domain and activated genes that have expression patterns inside the Lbx1-expressing domain., Conclusions/significance: Constraining epistasis analysis of Lbx1 to only those cells that normally express Lbx1 allowed unprecedented sensitivity in identifying Lbx1 network interactions and allowed the interactions to be assigned to a specific set of cell populations. We call this method ANCEA, or active node constrained epistasis analysis, and think that it will be generally useful in discovering and assigning network interactions to specific populations. We discuss how ANCEA, coupled with population partitioning analysis, can greatly facilitate the systematic dissection of transcriptional networks that underlie mammalian patterning.

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.

Association and relay neurons that are generated in the dorsal spinal cord play essential roles in transducing somatosensory information. During development, these two major neuronal classes are delineated by the expression of the homeodomain transcription factor Lbx1. Lbx1 is expressed in and required for the correct specification of three early dorsal interneuron populations and late-born neurons that form the substantia gelatinosa. In mice lacking Lbx1, cells types that arise in the ventral alar plate acquire more dorsal identities. This results in the loss of dorsal horn association interneurons, excess production of commissural neurons, and disrupted sensory afferent innervation of the dorsal horn. Lbx1, therefore, plays a critical role in the development of sensory pathways in the spinal cord that relay pain and touch.