Analysis of the evolution of resistance to multiple antibiotics enables prediction of the Escherichia coli phenotype-based fitness landscape.

The fitness landscape represents the complex relationship between genotype or phenotype and fitness under a given environment, the structure of which allows the explanation and prediction of evolutionary trajectories. Although previous studies have constructed fitness landscapes by comprehensively studying the mutations in specific genes, the high dimensionality of genotypic changes prevents us from developing a fitness landscape capable of predicting evolution for the whole cell. Herein, we address this problem by inferring the phenotype-based fitness landscape for antibiotic resistance evolution by quantifying the multidimensional phenotypic changes, i.e., time-series data of resistance for eight different drugs. We show that different peaks of the landscape correspond to different drug resistance mechanisms, thus supporting the validity of the inferred phenotype-fitness landscape. We further discuss how inferred phenotype-fitness landscapes could contribute to the prediction and control of evolution. This approach bridges the gap between phenotypic/genotypic changes and fitness while contributing to a better understanding of drug resistance evolution.

Emergence of dorsal-ventral polarity in ESC-derived retinal tissue.

We previously demonstrated that mouse embryonic stem cell (mESC)-derived retinal epithelium self-forms an optic cup-like structure. In the developing retina, the dorsal and ventral sides differ in terms of local gene expression and morphological features. This aspect has not yet been shown in vitro Here, we demonstrate that mESC-derived retinal tissue spontaneously acquires polarity reminiscent of the dorsal-ventral (D-V) patterning of the embryonic retina. Tbx5 and Vax2 were expressed in a mutually exclusive manner, as seen in vivo Three-dimensional morphometric analysis showed that the in vitro-formed optic cup often contains cleft structures resembling the embryonic optic fissure. To elucidate the mechanisms underlying the spontaneous D-V polarization of mESC-derived retina, we examined the effects of patterning factors, and found that endogenous BMP signaling plays a predominant role in the dorsal specification. Further analysis revealed that canonical Wnt signaling, which was spontaneously activated at the proximal region, acts upstream of BMP signaling for dorsal specification. These observations suggest that D-V polarity could be established within the self-formed retinal neuroepithelium by intrinsic mechanisms involving the spatiotemporal regulation of canonical Wnt and BMP signals.

Self-organizing optic-cup morphogenesis in three-dimensional culture.

Balanced organogenesis requires the orchestration of multiple cellular interactions to create the collective cell behaviours that progressively shape developing tissues. It is currently unclear how individual, localized parts are able to coordinate with each other to develop a whole organ shape. Here we report the dynamic, autonomous formation of the optic cup (retinal primordium) structure from a three-dimensional culture of mouse embryonic stem cell aggregates. Embryonic-stem-cell-derived retinal epithelium spontaneously formed hemispherical epithelial vesicles that became patterned along their proximal-distal axis. Whereas the proximal portion differentiated into mechanically rigid pigment epithelium, the flexible distal portion progressively folded inward to form a shape reminiscent of the embryonic optic cup, exhibited interkinetic nuclear migration and generated stratified neural retinal tissue, as seen in vivo. We demonstrate that optic-cup morphogenesis in this simple cell culture depends on an intrinsic self-organizing program involving stepwise and domain-specific regulation of local epithelial properties.

Intrinsic transition of embryonic stem-cell differentiation into neural progenitors.

The neural fate is generally considered to be the intrinsic direction of embryonic stem (ES) cell differentiation. However, little is known about the intracellular mechanism that leads undifferentiated cells to adopt the neural fate in the absence of extrinsic inductive signals. Here we show that the zinc-finger nuclear protein Zfp521 is essential and sufficient for driving the intrinsic neural differentiation of mouse ES cells. In the absence of the neural differentiation inhibitor BMP4, strong Zfp521 expression is intrinsically induced in differentiating ES cells. Forced expression of Zfp521 enables the neural conversion of ES cells even in the presence of BMP4. Conversely, in differentiation culture, Zfp521-depleted ES cells do not undergo neural conversion but tend to halt at the epiblast state. Zfp521 directly activates early neural genes by working with the co-activator p300. Thus, the transition of ES cell differentiation from the epiblast state into neuroectodermal progenitors specifically depends on the cell-intrinsic expression and activator function of Zfp521.

Subregional specification of embryonic stem cell-derived ventral telencephalic tissues by timed and combinatory treatment with extrinsic signals.

During early telencephalic development, the major portion of the ventral telencephalic (subpallial) region becomes subdivided into three regions, the lateral (LGE), medial (MGE), and caudal (CGE) ganglionic eminences. In this study, we systematically recapitulated subpallial patterning in mouse embryonic stem cell (ESC) cultures and investigated temporal and combinatory actions of patterning signals. In serum-free floating culture, the dorsal-ventral specification of ESC-derived telencephalic neuroectoderm is dose-dependently directed by Sonic hedgehog (Shh) signaling. Early Shh treatment, even before the expression onset of Foxg1 (also Bf1; earliest marker of the telencephalic lineage), is critical for efficiently generating LGE progenitors, and continuous Shh signaling until day 9 is necessary to commit these cells to the LGE lineage. When induced under these conditions and purified by fluorescence-activated cell sorter, telencephalic cells efficiently differentiated into Nolz1(+)/Ctip2(+) LGE neuronal precursors and subsequently, both in culture and after in vivo grafting, into DARPP32(+) medium-sized spiny neurons. Purified telencephalic progenitors treated with high doses of the Hedgehog (Hh) agonist SAG (Smoothened agonist) differentiated into MGE- and CGE-like tissues. Interestingly, in addition to strong Hh signaling, the efficient specification of MGE cells requires Fgf8 signaling but is inhibited by treatment with Fgf15/19. In contrast, CGE differentiation is promoted by Fgf15/19 but suppressed by Fgf8, suggesting that specific Fgf signals play different, critical roles in the positional specification of ESC-derived ventral subpallial tissues. We discuss a model of the antagonistic Fgf8 and Fgf15/19 signaling in rostral-caudal subpallial patterning and compare it with the roles of these molecules in cortical patterning.

Samurai in Japan: Class System-Related Morphological Differences in Maxillofacial Regions in the Edo Period.

Previous studies have reported that compared to commoners in Japan's Edo period, samurai had long heads, more dental irregularities, and slightly worn teeth. However, these studies did not measure the mandible or only measured length. Angular analysis is essential to evaluate the maxillofacial morphology, but there are no comparative studies of samurai and commoners. This study explored the differences in maxillofacial morphology between samurai and commoners in the Edo period. Thirty male skeletons (samurai) and thirty-eight male skeletons (commoners) were used as materials from the National Museum of Nature and Science. The selected specimens were adults aged between 20 and 59 years without serious skeletal damage and with stable occlusion of the molars. We used three-dimensional scanning to measure the specimens' skeletal, alveolar, and facial widths. The mandibular plane angle and the gonial angle were significantly larger in the samurai than in the commoners. The ratio of the intermandibular first molars, interzygomatic arch, and mandibular width was significantly shorter in the samurai than in the commoners. The samurai had a high angle tendency and smaller mandibular width than the commoners, reflecting the class system.

Development of a device that generates a temperature gradient in a microtiter plate for microbial culture.

Although temperature is a fundamental parameter in biology, testing various temperature conditions simultaneously is often difficult. In the present study, we developed a device for generating a temperature gradient in arrays of wells on a microtiter plate. This device consists of a pair of Peltier elements and temperature sensors placed on both ends of a flat aluminum bar to generate a linear temperature gradient. The device loads a microtiter plate at the center of the aluminum bar and transfers the temperature gradient to the bottom of the wells in the plate. This device successfully maintained a temperature gradient of 38.2 to 43.1°C on the horizontal axis of a 96-well microtiter plate in an incubator at 31°C. Furthermore, using this device, we demonstrated a laboratory evolution experiment of Escherichia coli, which was selected on the basis of its ability to grow at high temperatures. The developed device also facilitates a two-dimensional assay to determine the effects of temperature and drug concentrations on cellular growth.

Cv2, functioning as a pro-BMP factor via twisted gastrulation, is required for early development of nephron precursors.

The fine-tuning of BMP signals is critical for many aspects of complex organogenesis. In this report, we show that the augmentation of BMP signaling by a BMP-binding secreted factor, Crossveinless2 (Cv2), is essential for the early embryonic development of mammalian nephrons. In the Cv2-null mouse, the number of cap condensates (clusters of nephron progenitors, which normally express Cv2) was decreased, and the condensate cells exhibited a reduced level of aggregation. In these Cv2(-/-) condensates, the level of phosphorylated Smad1 (pSmad1) was substantially lowered. The loss of a Bmp7 allele in the Cv2(-/-) mouse enhanced the cap condensate defects and further decreased the level of pSmad1 in this tissue. These observations indicated that Cv2 has a pro-BMP function in early nephrogenesis. Interestingly, the renal defects of the Cv2(-/-) mutant were totally suppressed by a null mutation of Twisted gastrulation (Tsg), which encodes another BMP-binding factor, showing that Cv2 exerts its pro-BMP nephrogenic function Tsg-dependently. By using an embryonic kidney cell line, we presented experimental evidence showing that Cv2 enhances pro-BMP activity of Tsg. These findings revealed the molecular hierarchy between extracellular modifiers that orchestrate local BMP signal peaks in the organogenetic microenvironment.

Minimization of exogenous signals in ES cell culture induces rostral hypothalamic differentiation.

Embryonic stem (ES) cells differentiate into neuroectodermal progenitors when cultured as floating aggregates in serum-free conditions. Here, we show that strict removal of exogenous patterning factors during early differentiation steps induces efficient generation of rostral hypothalamic-like progenitors (Rax(+)/Six3(+)/Vax1(+)) in mouse ES cell-derived neuroectodermal cells. The use of growth factor-free chemically defined medium is critical and even the presence of exogenous insulin, which is commonly used in cell culture, strongly inhibits the differentiation via the Akt-dependent pathway. The ES cell-derived Rax(+) progenitors generate Otp(+)/Brn2(+) neuronal precursors (characteristic of rostral-dorsal hypothalamic neurons) and subsequently magnocellular vasopressinergic neurons that efficiently release the hormone upon stimulation. Differentiation markers of rostral-ventral hypothalamic precursors and neurons are induced from ES cell-derived Rax(+) progenitors by treatment with Shh. Thus, in the absence of exogenous growth factors in medium, the ES cell-derived neuroectodermal cells spontaneously differentiate into rostral (particularly rostral-dorsal) hypothalamic-like progenitors, which generate characteristic hypothalamic neuroendocrine neurons in a stepwise fashion, as observed in vivo. These findings indicate that, instead of the addition of inductive signals, minimization of exogenous patterning signaling plays a key role in rostral hypothalamic specification of neural progenitors derived from pluripotent cells.

Laboratory evolution of Mycobacterium on agar plates for analysis of resistance acquisition and drug sensitivity profiles.

Drug-resistant tuberculosis (TB) is a growing public health problem. There is an urgent need for information regarding cross-resistance and collateral sensitivity relationships among drugs and the genetic determinants of anti-TB drug resistance for developing strategies to suppress the emergence of drug-resistant pathogens. To identify mutations that confer resistance to anti-TB drugs in Mycobacterium species, we performed the laboratory evolution of nonpathogenic Mycobacterium smegmatis, which is closely related to Mycobacterium tuberculosis, against ten anti-TB drugs. Next, we performed whole-genome sequencing and quantified the resistance profiles of each drug-resistant strain against 24 drugs. We identified the genes with novel meropenem (MP) and linezolid (LZD) resistance-conferring mutation, which also have orthologs, in M. tuberculosis H37Rv. Among the 240 possible drug combinations, we identified 24 pairs that confer cross-resistance and 18 pairs that confer collateral sensitivity. The acquisition of bedaquiline or linezolid resistance resulted in collateral sensitivity to several drugs, while the acquisition of MP resistance led to multidrug resistance. The MP-evolved strains showed cross-resistance to rifampicin and clarithromycin owing to the acquisition of a mutation in the intergenic region of the Rv2864c ortholog, which encodes a penicillin-binding protein, at an early stage. These results provide a new insight to tackle drug-resistant TB.

Mutational property of newly identified mutagen l-glutamic acid γ-hydrazide in Escherichia coli.

We previously found that an l-glutamine analog l-glutamic acid γ-hydrazide has high mutagenic activity through the high-throughput laboratory evolution of Escherichia coli. In this study, mutagenicity and mutational property of l-glutamic acid γ-hydrazide were examined by the Ames test and mutation accumulation experiments using E. coli. The Ames test revealed that l-glutamic acid γ-hydrazide showed higher mutagenic activity without metabolic activation than known mutagens 2-aminoanthracene, and cobalt(II) acetate tetrahydrate. This result indicates that l-glutamic acid γ-hydrazide does not require metabolic activation for mutagenic activity in E. coli. Mutation accumulation experiments and whole-genome sequencing analysis revealed the number and spectrum of the accumulated mutations with or without l-glutamic acid γ-hydrazide. In the presence of l-glutamic acid γ-hydrazide, MDS42 strain accumulated 392.3 ± 116.2 point mutations during 30 passages corresponding to 777 generations, while MDS42 strain accumulated 1.5 ± 2.5 point mutations without l-glutamic acid γ-hydrazide during 50 passages corresponding to 1341 generations. The mutational spectrum of l-glutamic acid γ-hydrazide was G/C to A/T transition (82.2 ± 4.3 %) and A/T to G/C transition (17.4 ± 4.3 %). These results indicated that l-glutamic acid γ-hydrazide has a strong mutagenic activity.

High-throughput laboratory evolution reveals evolutionary constraints in Escherichia coli.

Understanding the constraints that shape the evolution of antibiotic resistance is critical for predicting and controlling drug resistance. Despite its importance, however, a systematic investigation of evolutionary constraints is lacking. Here, we perform a high-throughput laboratory evolution of Escherichia coli under the addition of 95 antibacterial chemicals and quantified the transcriptome, resistance, and genomic profiles for the evolved strains. Utilizing machine learning techniques, we analyze the phenotype-genotype data and identified low dimensional phenotypic states among the evolved strains. Further analysis reveals the underlying biological processes responsible for these distinct states, leading to the identification of trade-off relationships associated with drug resistance. We also report a decelerated evolution of ß-lactam resistance, a phenomenon experienced by certain strains under various stresses resulting in higher acquired resistance to ß-lactams compared to strains directly selected by ß-lactams. These findings bridge the genotypic, gene expression, and drug resistance gap, while contributing to a better understanding of evolutionary constraints for antibiotic resistance.

Complete Genome Sequence of a Radioresistant Bacterial Strain, Deinococcus grandis ATCC 43672.

Deinococcus grandis is a radioresistant bacterial species isolated from freshwater fish. In this article, we report the complete genome sequence of D. grandis strain ATCC 43672. This sequence is useful for comparative genomics to understand the traits of Deinococcus species and can be used as a reference in experimental genetics.

Complete Genome Sequences of Three Star-Shaped Bacteria, Stella humosa, Stella vacuolata, and Stella Species ATCC 35155.

Stella species are unique star-shaped alphaproteobacteria found in various environments. We report the complete genome sequences of three Stella strains, Stella humosa ATCC 43930, Stella vacuolata ATCC 43931, and Stella species ATCC 35155. These are the first complete genome sequences of members of the genus Stella.

Functional analysis of chick ONT1 reveals distinguishable activities among olfactomedin-related signaling factors.

The Olfactomedin family is a relatively new class of extracellular proteins. Two family members have been shown to play roles in the early development of ectodermal tissues: Noelin enhances neural crest generation in chick and Tiarin promotes dorsal neural specification in Xenopus. In this study, we introduce a novel member of the Olfactomedin family, ONT1. In the early chick embryo, ONT1 expression first appears at Hensen's node and subsequently in the axial and paraxial mesoderm. When the neural tube closes, strong expression of ONT1 is transiently found in the roof plate region from the rostral midbrain to the hindbrain. Overexpression of ONT1 in these regions prolongs the generation of neural crest cells in a manner similar to that of Noelin. Interestingly, ONT1 and Noelin have opposing effects on the expression of the migrating neural crest marker HNK-1 in the chick: they, respectively, cause suppression and ectopic induction of this marker. Differential activities among Olfactomedin-related factors are further examined in Xenopus. Microinjection of ONT1 mRNA into the Xenopus embryo expands the expression domain of the neural crest marker FoxD3 at the neurula stage whereas overexpression of Tiarin or Noelin suppresses FoxD3. ONT1 exhibits no dorsalizing effects on the Xenopus neural tube, which contrasts with the strong dorsalizing activity seen for Tiarin. Thus, distinct Olfactomedin-related factors evoke qualitatively different phenotypes even in the same experimental systems, suggesting that Olfactomedin family uses multiple response systems to mediate its signals in embryogenesis.

Gene disruption/knock-in analysis of mONT3: vector construction by employing both in vivo and in vitro recombinations.

We report the isolation, spatial/temporal expression and gene disruption phenotype of the mouse ONT3 (mONT3) gene, which encodes a novel secreted signaling protein belonging to the Olfactomedin/Noelin/Tiarin family. During early embryogenesis, mONT3 is detected in the proximal region of the allantois on embryonic day (E) 7.25, in the lateral plate mesoderm on E 8.0 and in the CNS and heart on E 8.5. The homozygous mutant is born normal and fertile. For the expression pattern and loss-of-function analyses, we have successfully generated the LacZ-knock-in targeting vector directly from BACs carrying mouse genomic fragments by combining in vivo and in vitro recombination techniques. This approach enables rapid and reproducible construction of the fully functional vectors within two weeks without the use of restriction enzyme digestion and ligation, or the use of PCR-amplification of large genomic fragments. In addition, this method is applicable to rapid generation of transgenic vectors, demonstrating its versatility in reverse genetic studies.

Self-formation of optic cups and storable stratified neural retina from human ESCs.

In this report, we demonstrate that an optic cup structure can form by self-organization in human ESC culture. The human ESC-derived optic cup is much larger than the mouse ESC-derived one, presumably reflecting the species differences. The neural retina in human ESC culture is thick and spontaneously curves in an apically convex manner, which is not seen in mouse ESC culture. In addition, human ESC-derived neural retina grows into multilayered tissue containing both rods and cones, whereas cone differentiation is rare in mouse ESC culture. The accumulation of photoreceptors in human ESC culture can be greatly accelerated by Notch inhibition. In addition, we show that an optimized vitrification method enables en bloc cryopreservation of stratified neural retina of human origin. This storage method at an intermediate step during the time-consuming differentiation process provides a versatile solution for quality control in large-scale preparation of clinical-grade retinal tissues.

Twisted gastrulation mutation suppresses skeletal defect phenotypes in Crossveinless 2 mutant mice.

Bone morphogenetic protein (BMP) signaling controls various aspects of organogenesis, including skeletal development. We previously demonstrated that the pro-BMP function of Crossveinless 2 (Cv2) is required for axial and non-axial skeletal development in mice. Here, we showed that skeletal defects in the Cv2-null mutant were reversed by the additional deletion of Twisted gastrulation (Tsg). Whereas the Cv2(-/-) mutant lacks a substantial portion of the lumbar vertebral arches, Cv2(-/-);Tsg(-/-) mice have almost normal arches. Suppression of Cv2(-/-) phenotypes is also seen in the non-axial skeleton, including the ribs, humerus, skull, and laryngeal and tracheal cartilages. In contrast, the Tsg(-/-) phenotype in the head is not significantly affected by the Cv2 mutation. These findings demonstrate that Tsg mutation is epistatic to Cv2 mutation in the major skeletal phenotypes, suggesting that the pro-BMP activity of Cv2 is, at least in part, dependent on Tsg. We also present genetic evidence for the context-dependent functional relationship between Tsg and Cv2 during mouse development.

Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.

Here, we demonstrate self-organized formation of apico-basally polarized cortical tissues from ESCs using an efficient three-dimensional aggregation culture (SFEBq culture). The generated cortical neurons are functional, transplantable, and capable of forming proper long-range connections in vivo and in vitro. The regional identity of the generated pallial tissues can be selectively controlled (into olfactory bulb, rostral and caudal cortices, hem, and choroid plexus) by secreted patterning factors such as Fgf, Wnt, and BMP. In addition, the in vivo-mimicking birth order of distinct cortical neurons permits the selective generation of particular layer-specific neurons by timed induction of cell-cycle exit. Importantly, cortical tissues generated from mouse and human ESCs form a self-organized structure that includes four distinct zones (ventricular, early and late cortical-plate, and Cajal-Retzius cell zones) along the apico-basal direction. Thus, spatial and temporal aspects of early corticogenesis are recapitulated and can be manipulated in this ESC culture.

Essential pro-Bmp roles of crossveinless 2 in mouse organogenesis.

We here report essential roles of the Bmp-binding protein crossveinless 2 (Cv2; Bmper) in mouse organogenesis. In the null Cv2 mutant mouse, gastrulation occurs normally, but a number of defects are found in Cv2-expressing tissues such as the skeleton. Cartilage differentiation by Bmp4 treatment is reduced in cultured Cv2(-/-) fibroblasts. Moreover, the defects in the vertebral column and eyes of the Cv2(-/-) mouse are substantially enhanced by deleting one copy of the Bmp4 gene, suggesting a pro-Bmp role of Cv2 in the development of these organs. In addition, the Cv2(-/-) mutant exhibits substantial defects in Bmp-dependent processes of internal organ formation, such as nephron generation in the kidney. This kidney hypoplasia is synergistically enhanced by the additional deletion of Kcp (Crim2) which encodes a pro-Bmp protein structurally related to Cv2. This study demonstrates essential pro-Bmp functions of Cv2 for locally restricted signal enhancement in multiple aspects of mammalian organogenesis.