Stomata are microscopic valves on the plant epidermis that played a critical role in the evolution of land plants. Studies in the model dicot Arabidopsis thaliana have identified key transcription factors and signaling pathways controlling stomatal patterning and differentiation. Three paralogous Arabidopsis basic helix-loop-helix proteins, SPEECHLESS (SPCH), MUTE, and FAMA, mediate sequential steps of cell-state transitions together with their heterodimeric partners SCREAM (SCRM) and SCRM2. Cell-cell signaling components, including putative ligands, putative receptors, and mitogen-activated protein kinase cascades, orient asymmetric cell divisions and prevent overproduction and clustering of stomata. The recent availability of genome sequence and reverse genetics tools for model monocots and basal land plants allows for the examination of the conservation of genes important in stomatal patterning and differentiation. Studies in grasses have revealed that divergence of SPCH-MUTE-FAMA predates the evolutionary split of monocots and dicots and that these proteins show conserved and novel roles in stomatal differentiation. By contrast, specific asymmetric cell divisions in Arabidopsis and grasses require unique molecular components. Molecular phylogenetic analysis implies potential conservation of signaling pathways and prototypical functions of the transcription factors specifying stomatal differentiation.

Stomata, valves on the plant epidermis, are critical for plant growth and survival, and the presence of stomata impacts the global water and carbon cycle. Although transcription factors and cell-cell signaling components regulating stomatal development have been identified, it remains unclear as to how their regulatory interactions are translated into two-dimensional patterns of stomatal initial cells. Using molecular genetics, imaging, and mathematical simulation, we report a regulatory circuit that initiates the stomatal cell-lineage. The circuit includes a positive feedback loop constituting self-activation of SCREAMs that requires SPEECHLESS. This transcription factor module directly binds to the promoters and activates a secreted signal, EPIDERMAL PATTERNING FACTOR2, and the receptor modifier TOO MANY MOUTHS, while the receptor ERECTA lies outside of this module. This in turn inhibits SPCH, and hence SCRMs, thus constituting a negative feedback loop. Our mathematical model accurately predicts all known stomatal phenotypes with the inclusion of two additional components to the circuit: an EPF2-independent negative-feedback loop and a signal that lies outside of the SPCH•SCRM module. Our work reveals the intricate molecular framework governing self-organizing two-dimensional patterning in the plant epidermis.

Chordates evolved a unique body plan within deuterostomes and are considered to share five morphological characters, a muscular postanal tail, a notochord, a dorsal neural tube, an endostyle, and pharyngeal gill slits. The phylum Chordata typically includes three subphyla, Cephalochordata, Vertebrata, and Tunicata, the last showing a chordate body plan only as a larva. Hemichordates, in contrast, have pharyngeal gill slits, an endostyle, and a postanal tail but appear to lack a notochord and dorsal neural tube. Because hemichordates are the sister group of echinoderms, the morphological features shared with the chordates must have been present in the deuterostome ancestor. No extant echinoderms share any of the chordate features, so presumably they have lost these structures evolutionarily. We review the development of chordate characters in hemichordates and present new data characterizing the pharyngeal gill slits and their cartilaginous gill bars. We show that hemichordate gill bars contain collagen and proteoglycans but are acellular. Hemichordates and cephalochordates, or lancelets, show strong similarities in their gill bars, suggesting that an acellular cartilage may have preceded cellular cartilage in deuterostomes. Our evidence suggests that the deuterostome ancestor was a benthic worm with gill slits and acellular gill cartilages.

No abstract

Deuterostomes are a monophyletic group of an imals con tai ni n g vertebrates, lancelets, tunicates, hemichordates, ech inoderms, and xenoturbellids. F our out of these six extant groups-vertebrates, la ncelets, tunicates, and h emichordates-have pharyngeal gill slits. All groups of deuterostome an im als that have pharyngeal gill slit s also have a pharyn geal sk eleton supporting the pharyngeal ope nin gs, except tunicates. We previously found that pharyngeal cartilage in hemichordates and ceph aloch ordat es contains a fibrillar collagen protein similar to vertebrate type II collagen, but unlike vertebrate cartilage, the in vertebrate deuterostome cartilages ar e acellular. We found SaxE and fibrillar collagen expression in the pharyngeal endodermal cells adjacen t to where the cartilages form . These same endodermal epithelial cells also express Pax.l /9 , a marker of pharyngeal endoderm in vertebrates, lancelets, tunicates, and hemichordates. In situ exp eriments with a cephalochordate fibrillar collagen al so showed expression in pharyn geal en dode rm, as well as the ectoderm a nd the mesodermal coelomic pou ches lining the gill bars. These results indicate that the pharyngeal en doderm al cells are re sp on sible for secr etion of the cartilage in hemichordates, wherea s in lancelets, all the pharyngeal cells su r r ou n din g the gill bars, ect odermal, endodermal, and mesodermal may be resp onsible for cartilage formation . We propose that endoderm secretion was primarily the ancestral mode of making pharyngeal cartilages in deutero stomes. Later the evolu ti on ary origin of neural crest allow ed co-opti on of the gene n etwork for th e secretion of pharyngeal cartilage matrix in the new migratory n eural cr est cell populations found in vertebrat es.

Ptychodera flava is a hemichordate whose anterior structures regenerate reproducibly from posterior trunk pieces when amputated. We characterized the cellular processes of anterior regeneration with respect to programmed cell death and cell proliferation, after wound healing. We found scattered proliferating cells at day 2 of regeneration using a proliferating cell nuclear antigen antibody. On day 4, most proliferating cells were associated with the nerve tract under the epidermis, and on day 6, a small proboscis derived from proliferated cells was regenerated, and a mouth had broken though the epidermis. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick end-labeling) detected elevated levels of apoptosis in the endoderm that began furthest away from the region of wound healing, then moved anteriorly over 8 days. Posterior to anterior apoptosis is likely to remove digestive endoderm for later differentiation of pharyngeal endoderm. We hypothesize that P. flava regeneration is nerve dependent and that remodeling in the gut endoderm plays an important role in regeneration.

No abstract

Stomata are an essential land plant innovation whose patterning and density are under genetic and environmental control. Recently, several putative ligands have been discovered that influence stomatal density, and they all belong to the epidermal patterning factor-like family of secreted cysteine-rich peptides. Two of these putative ligands, EPF1 and EPF2, are expressed exclusively in the stomatal lineage cells and negatively regulate stomatal density. A third, EPFL6 or CHALLAH, is also a negative regulator of density, but is expressed subepidermally in the hypocotyl. A fourth, EPFL9 or STOMAGEN, is expressed in the mesophyll tissues and is a positive regulator of density. Genetic evidence suggests that these ligands may compete for the same receptor complex. Proper stomatal patterning is likely to be an intricate process involving ligand competition, regional specificity, and communication between tissue layers. EPFL-family genes exist in the moss Physcomitrella patens, the lycophyte Selaginella moellendorffii, and rice, Oryza sativa, and their sequence analysis yields several genes some of which are related to EPF1, EPF2, EPFL6, and EPFL9. Presence of these EPFL family members in the basal land plants suggests an exciting hypothesis that the genetic components for stomatal patterning originated early in land plant evolution.