1. Volume 28, Issue 3, Pages 479-486.e5 (February 2018)
Generative Cell Specification Requires Transcription Factors Evolutionarily Conserved in Land Plants 
Shohei Yamaoka, Ryuichi Nishihama, Yoshihiro Yoshitake, Sakiko Ishida, Keisuke Inoue, Misaki Saito, Keitaro Okahashi, Haonan Bao, Hiroyuki Nishida, Katsushi Yamaguchi, Shuji Shigenobu, Kimitsune Ishizaki, Katsuyuki T. Yamato, Takayuki Kohchi 
Current Biology 
Volume 28, Issue 3, Pages e5 (February 2018)
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2. Current Biology 2018 28, 479-486.e5DOI: (10.1016/j.cub.2017.12.053)
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3. Figure 1 Germ Cell Differentiation in M. polymorpha and A. thaliana
(A) M. polymorpha male initial cell develops into antheridium, where spermatogenous cells (SCs) repeat mitotic divisions to form multiple sperm mother cells (SMCs), each of which undergoes diagonal division and cell morphogenesis to form two flagellated sperm [7–10].
(B) M. polymorpha female initial cell develops into archegonium, whose central cells divide anticlinally to form egg, ventral canal cell (VC), and neck canal cells (NCs). VC and NCs further degenerate to form a path to egg for fertilization [7–9].
(C) In A. thaliana, single-cell microspore divides asymmetrically and differentiates GC, which is then internalized into vegetative cell cytoplasm and divides symmetrically to produce two sperm cells.
Green circles indicate nuclear BNB accumulation (see also Figure 3).
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4. Figure 2 MpBNB Controls Gametangiophore Development in M. polymorpha
(A) Structures of the rearranged region in WT and hpt2040 genomes. Magenta arrowheads and blue vertical bars indicate MpBNB gene and primer pair positions used in linkage analysis (shown in B), respectively.
(B) Linkage analysis. Primer pair positions are shown in (A).
(C) MpBNB gene structure. Black box indicates CDS.
(D) 5-week-old 35S-MpBNB plants grown under FR-lacking light conditions. Arrowheads indicate gametangiophores.
(E) 25-day-old MpBNB-GR plants grown under FR-lacking light conditions in the presence and absence of DEX. Images in dotted squares are magnified in insets.
(F) 60-day-old WT and Mpbnbko plants grown under FR-enriched light conditions. Images in dotted squares are magnified in insets.
(G) Timing of gametangiophore formation. Data represent mean + SD of days until first immature gametangiophore appeared at thallus tip in three independent experiments (n ≥ 16).
Scale bars, 5 mm (D) and 1 cm (5 mm in insets) (E and F). See also Table S1 for RNA-seq analysis, Figure S1 for gene phylogeny and bHLH domain structures, and Figure S2 for generation of Mpbnbko plants.
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5. Figure 3
BNB Accumulates at an Early Stage of Germ Cell Differentiation in M. polymorpha and A. thaliana Gametophytes
(A) Bright-field, single confocal, and merged images of initial cells and immature gametangia in MpBNB-Citrine plants. Arrows and arrowheads indicate egg and VC nuclei, respectively. Dotted lines enclose eggs, SCs in early immature antheridium, and SMCs in late immature antheridium.
(B) Single confocal images of DAPI-stained pollen from GFP-BNB2 plants. Open arrowheads, closed arrowheads, and arrows indicate nuclei of uninucleate pollen, smaller cells of binucleate pollen, and sperm cells, respectively.
Scale bars, 10 μm (initial cells and early immature gametangia), 50 μm (late immature gametantia) (A), and 10 μm (B). See also Figure S2 for generation of MpBNB-Citrine plants, Figure S3 for observation of MpBNB-Citrine during gametangiophore development, and Figure S4 for growth phenotype of GFP-BNB1/2 plants.
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6. Figure 4
BNB1 and BNB2 Are Redundantly Required for GC Specification in A. thaliana
(A) Fluorescent image of DAPI-stained pollen from bnb1−/− bnb2+/− plant.
(B) Pollen phenotype. Data represent mean ± SD of percentages of pollen grains counted in three to five independent experiments (n = 34–344).
(C) TEM images of WT and bnb1 bnb2 pollen. Images in dotted squares are magnified on right. vn, vegetative nucleus; sc, sperm cell; vl, vegetative-like nucleus.
(D) Fluorescent images of DAPI-stained pollen from WT and bnb1+/− bnb2−/− plants.
(E) Phenotype of developing pollen from WT and bnb1+/− bnb2−/− plants. Data represent percentages of pollen grains from flower buds serially isolated from independent inflorescences.
(F) Single confocal images of DAPI-stained pollen from bnb1−/− bnb2+/− plants carrying H3.10-RFP. Arrows and closed and open arrowheads indicate nuclei of sperm cells, GCs, and mutant cells, respectively.
Scale bars, 10 μm (A, D, and F) and 5 μm (1 μm in magnified images) (C). See also Figure S1 for gene phylogeny and bHLH domain structures and Figure S4 and Table S2 for identification and characterization of mutant plants.
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