The Impact of Lactobacillus Probiotics on the Gut Microbiota in Children With Short Bowel Syndrome.

BACKGROUND: Children with short bowel syndrome (SBS) frequently struggle with malabsorption and poor growth. The intestinal microbiota plays an important role in gut function, and children with SBS have known deficiencies in some commensal gut microbes. One strategy to enhance the gut microbiota is by taking probiotics. However, the efficacy of this approach is not well established. We hypothesized that probiotic supplementation would result in increased levels of the supplemented bacteria and improved growth. MATERIALS AND METHODS: Children with SBS who had weaned from parenteral nutrition but with suboptimal growth were randomized to receive probiotics (Lactobacillus rhamnosus and Lactobacillus johnsonii) or placebo daily for 2 mo. The gut microbiota from monthly stool samples were compared between groups using 16S ribosomal ribonucleic acid sequencing and quantitative polymerase chain reaction. Growth between groups was also compared. Statistical analysis was completed using Mann-Whitney, Kruskal-Wallis, and chi-square tests as appropriate. RESULTS: Eighteen children with SBS completed the study (n = 9 per group). There were no significant changes to the major bacterial families in either group. Median relative abundance of Lactobacillus did not differ between groups at baseline or at the end of the study (7.67 versus 13.23, P = 0.523 and 1.93 versus 15.8, P = 0.161). Median z scores for weight and length did not differ between groups at the beginning or end of the study. CONCLUSIONS: The efficacy of daily probiotic use in children with intestinal failure is unknown. In this study, Lactobacillus probiotics did not result in a predictable change to the fecal microbiota or overall growth compared with placebo in these patients.

The effects of lateral line ablation and regeneration in schooling giant danios.

Fish use multiple sensory systems, including vision and their lateral line system, to maintain position and speed within a school. Although previous studies have shown that ablating the lateral line alters schooling behavior, no one has examined how the behavior recovers as the sensory system regenerates. We studied how schooling behavior changes in giant danios, Devario aequipinnatus, when their lateral line system is chemically ablated and after the sensory hair cells regenerate. We found that fish could school normally immediately after chemical ablation, but that they had trouble schooling 1-2 weeks after the chemical treatment, when the hair cells had fully regenerated. We filmed groups of giant danios with two high-speed cameras and reconstructed the three-dimensional positions of each fish within a group. One fish in the school was treated with gentamycin to ablate all hair cells. Both types of neuromasts (canal and superficial) were completely ablated after treatment, but fully regenerated after 1 week. We quantified the structure of the school using nearest neighbor distance, bearing, elevation, and the cross-correlation of velocity between each pair of fish. Treated fish maintained a normal position within the school immediately after the lateral line ablation, but could not school normally 1 or 2 weeks after treatment, even though the neuromasts had fully regenerated. By 4-8 weeks post-treatment, the treated fish could again school normally. These results demonstrate that the behavioral recovery after lateral line ablation is a longer process than the regeneration of the hair cells themselves.

Antibiotic-Induced Depletion of Anti-inflammatory Clostridia Is Associated with the Development of Graft-versus-Host Disease in Pediatric Stem Cell Transplantation Patients.

Adult stem cell transplantation (SCT) patients with graft-versus-host-disease (GVHD) exhibit significant disruptions in gut microbial communities. These changes are associated with higher overall mortality and appear to be driven by specific antibiotic therapies. It is unclear whether pediatric SCT patients who develop GVHD exhibit similar antibiotic-induced gut microbiota community changes. Here, we show that pediatric SCT patients (from Children's Medical Center Dallas, n = 8, and Cincinnati Children's Hospital, n = 7) who developed GVHD showed a significant decline, up to 10-log fold, in gut anti-inflammatory Clostridia (AIC) compared with those without GVHD. In fact, the development of GVHD is significantly associated with this AIC decline and with cumulative antibiotic exposure, particularly antibiotics effective against anaerobic bacteria (P = .003, Firth logistic regression analysis). Using metagenomic shotgun sequencing analysis, we were able to identify specific commensal bacterial species, including AIC, that were significantly depleted in GVHD patients. We then used a preclinical GVHD model to verify our clinical observations. Clindamycin depleted AIC and exacerbated GVHD in mice, whereas oral AIC supplementation increased gut AIC levels and mitigated GVHD in mice. Together, these data suggest that an antibiotic-induced AIC depletion in the gut microbiota is associated with the development of GVHD in pediatric SCT patients.

Candida albicans Inhibits Pseudomonas aeruginosa Virulence through Suppression of Pyochelin and Pyoverdine Biosynthesis.

Bacterial-fungal interactions have important physiologic and medical ramifications, but the mechanisms of these interactions are poorly understood. The gut is host to trillions of microorganisms, and bacterial-fungal interactions are likely to be important. Using a neutropenic mouse model of microbial gastrointestinal colonization and dissemination, we show that the fungus Candida albicans inhibits the virulence of the bacterium Pseudomonas aeruginosa by inhibiting P. aeruginosa pyochelin and pyoverdine gene expression, which plays a critical role in iron acquisition and virulence. Accordingly, deletion of both P. aeruginosa pyochelin and pyoverdine genes attenuates P. aeruginosa virulence. Heat-killed C. albicans has no effect on P. aeruginosa, whereas C. albicans secreted proteins directly suppress P. aeruginosa pyoverdine and pyochelin expression and inhibit P. aeruginosa virulence in mice. Interestingly, suppression or deletion of pyochelin and pyoverdine genes has no effect on P. aeruginosa's ability to colonize the GI tract but does decrease P. aeruginosa's cytotoxic effect on cultured colonocytes. Finally, oral iron supplementation restores P. aeruginosa virulence in P. aeruginosa and C. albicans colonized mice. Together, our findings provide insight into how a bacterial-fungal interaction can modulate bacterial virulence in the intestine. Previously described bacterial-fungal antagonistic interactions have focused on growth inhibition or colonization inhibition/modulation, yet here we describe a novel observation of fungal-inhibition of bacterial effectors critical for virulence but not important for colonization. These findings validate the use of a mammalian model system to explore the complexities of polymicrobial, polykingdom infections in order to identify new therapeutic targets for preventing microbial disease.

Assessment of Distinct Gut Microbiome Signatures in a Diverse Cohort of Patients Undergoing Definitive Treatment for Rectal Cancer.

Introduction: Disparities in incidence and outcome of rectal cancer are multifactorial in etiology but may be due, in part, to differences in gut microbiome composition. We used serial robust statistical approaches to assess baseline gut microbiome composition in a diverse cohort of patients with rectal cancer receiving definitive treatment. Methods: Microbiome composition was compared by age at diagnosis (< 50 vs ≥ 50 years), race and ethnicity (White Hispanic vs non-Hispanic), and response to therapy. Alpha diversity was assessed using the Shannon, Chao1, and Simpson diversity measures. Beta diversity was explored using both Bray-Curtis dissimilarity and Aitchison distance with principal coordinate analysis. To minimize false-positive findings, we used two distinct methods for differential abundance testing: LinDA and MaAsLin2 (all statistics two-sided, Benjamini-Hochberg corrected false discovery rate < 0.05). Results: Among 64 patients (47% White Hispanic) with median age 51 years, beta diversity metrics showed significant clustering by race and ethnicity (p < 0.001 by both metrics) and by onset (Aitchison p = 0.022, Bray-Curtis p = 0.035). White Hispanic patients had enrichment of bacterial family Prevotellaceae (LinDA fold change 5.32, MaAsLin2 fold change 5.11, combined adjusted p = 0.0007). No significant differences in microbiome composition were associated with neoadjuvant therapy response. Conclusion: We identified distinct gut microbiome signatures associated with race and ethnicity and age of onset in a diverse cohort of patients undergoing definitive treatment for rectal cancer.

Susceptible bacteria can survive antibiotic treatment in the mammalian gastrointestinal tract without evolving resistance.

Antibiotic resistance and evasion are incompletely understood and complicated by the fact that murine interval dosing models do not fully recapitulate antibiotic pharmacokinetics in humans. To better understand how gastrointestinal bacteria respond to antibiotics, we colonized germ-free mice with a pan-susceptible genetically barcoded Escherichia coli clinical isolate and administered the antibiotic cefepime via programmable subcutaneous pumps, allowing closer emulation of human parenteral antibiotic dynamics. E. coli was only recovered from intestinal tissue, where cefepime concentrations were still inhibitory. Strikingly, "some" E. coli isolates were not cefepime resistant but acquired mutations in genes involved in polysaccharide capsular synthesis increasing their invasion and survival within human intestinal cells. Deleting wbaP involved in capsular polysaccharide synthesis mimicked this phenotype, allowing increased invasion of colonocytes where cefepime concentrations were reduced. Additionally, "some" mutant strains exhibited a persister phenotype upon further cefepime exposure. This work uncovers a mechanism allowing "select" gastrointestinal bacteria to evade antibiotic treatment.

Immune checkpoint blockade induces gut microbiota translocation that augments extraintestinal antitumor immunity.

Gut microbiota, specifically gut bacteria, are critical for effective immune checkpoint blockade therapy (ICT) for cancer. The mechanisms by which gut microbiota augment extraintestinal anticancer immune responses, however, are largely unknown. Here, we find that ICT induces the translocation of specific endogenous gut bacteria into secondary lymphoid organs and subcutaneous melanoma tumors. Mechanistically, ICT induces lymph node remodeling and dendritic cell (DC) activation, which facilitates the translocation of a selective subset of gut bacteria to extraintestinal tissues to promote optimal antitumor T cell responses in both the tumor-draining lymph nodes (TDLNs) and the primary tumor. Antibiotic treatment results in decreased gut microbiota translocation into mesenteric lymph nodes (MLNs) and TDLNs, diminished DC and effector CD8+ T cell responses, and attenuated responses to ICT. Our findings illuminate a key mechanism by which gut microbiota promote extraintestinal anticancer immunity.

Susceptible bacteria survive antibiotic treatment in the mammalian gastrointestinal tract without evolving resistance.

In vitro systems have provided great insight into the mechanisms of antibiotic resistance. Yet, in vitro approaches cannot reflect the full complexity of what transpires within a host. As the mammalian gut is host to trillions of resident bacteria and thus a potential breeding ground for antibiotic resistance, we sought to better understand how gut bacteria respond to antibiotic treatment in vivo . Here, we colonized germ-free mice with a genetically barcoded antibiotic pan-susceptible Escherichia coli clinical isolate and then administered the antibiotic cefepime via programmable subcutaneous pumps which allowed for closer emulation of human parenteral antibiotic pharmacokinetics/dynamics. After seven days of antibiotics, we were unable to culture E. coli from feces. We were, however, able to recover barcoded E. coli from harvested gastrointestinal (GI) tissue, despite high GI tract and plasma cefepime concentrations. Strikingly, these E. coli isolates were not resistant to cefepime but had acquired mutations â€" most notably in the wbaP gene, which encodes an enzyme required for the initiation of the synthesis of the polysaccharide capsule and lipopolysaccharide O antigen - that increased their ability to invade and survive within intestinal cells, including cultured human colonocytes. Further, these E. coli mutants exhibited a persister phenotype when exposed to cefepime, allowing for greater survival to pulses of cefepime treatment when compared to the wildtype strain. Our findings highlight a mechanism by which bacteria in the gastrointestinal tract can adapt to antibiotic treatment by increasing their ability to persist during antibiotic treatment and invade intestinal epithelial cells where antibiotic concentrations are substantially reduced.

Potential diagnostic biomarkers for pulmonary tuberculosis in humans are not elevated in Mycobacterium tuberculosis culture-positive Asian elephants (Elephas maximus).

OBJECTIVE: To determine (1) if chemokine (C-X-C motif) ligand 1 (CXCL1), matrix metalloproteinase 8 (MMP8), interleukin-10 (IL-10), interferon-γ (IFN-γ), and tumor necrosis factor-α (TNF-α) can be detected in serum from Asian elephants, and (2) if their concentrations are significantly elevated in Mycobacterium tuberculosis (M.tb) culture-positive elephants compared to -negative elephants. CXCL1, MMP8, IL-10, IFN-γ, and TNF-α were recently identified as potential diagnostic biomarkers for pulmonary tuberculosis in experimental studies in animals and humans. Therefore, we hypothesized that they would be detectable and significantly elevated in M.tb culture-positive elephants compared to M.tb culture-negative elephants. SAMPLE: 101 Asian elephant serum samples, including 91 samples from 6 M.tb-negative elephants and 10 samples from 5 M.tb-positive elephants (none of which exhibited clinical signs of disease). M.tb status was determined by trunk wash culture. PROCEDURES: Commercially available ELISA kits were used to determine the concentrations of each biomarker in serum samples. RESULTS: Biomarker concentrations were below the limit of detection for the assay in 100/101 (99%) samples for CXCL1, 98/101 (97%) samples for MMP8, 85/101 (84%) samples for IL-10, 75/101 (74%) samples for IFN-γ, and 45/101 (45%) samples for TNF-α. Multiple M.tb culture-positive elephants did not have detectable levels of any of the 5 biomarkers. CLINICAL RELEVANCE: CXCL1, MMP8, IL-10, IFN-γ, and TNF-α were not elevated in M.tb culture-positive Asian elephants compared to M.tb culture-negative Asian elephants. This may be related to disease state (ie, clinically asymptomatic). More sensitive assays are needed to better understand the role of these biomarkers in M.tb infection in Asian elephants.

A comparison of small bowel and fecal microbiota in children with short bowel syndrome.

BACKGROUND: Babies with short bowel syndrome (SBS) have small intestinal microbial disturbances that impact gut function. Characterizing the small bowel microbiota is challenging, and the utility of sampling stool is unclear. This study compares the microbiota from fecal samples and the small bowel. METHODS: Stool samples were collected (2016-2017) from infants with SBS and colon in continuity (COLON) or SBS with small bowel ostomy (sbSTOMA). The abundance and quantity of major bacterial genera was compared between groups and to healthy controls using 16S rRNA sequencing and qPCR. Kruskall-Wallis test was used for analysis with P values <0.05 considered significant. RESULTS: Samples (n = 41) were collected from 15 SBS infants (<2 years) (9 sbSTOMA, 6 COLON) and 3 healthy infants. Demographics and small intestinal length did not differ between sbSTOMA and COLON infants. The microbiota of SBS groups differed significantly from healthy controls. Fecal samples contained higher quantities of bacteria, but there were no significant differences between sbSTOMA and COLON groups in the abundance of facultative or obligate anaerobes, anti-inflammatory Clostridia, Enterobacteriaceae, or Bifidobacterium. CONCLUSION: Infants with SBS have disturbances to their intestinal microbiota. Sampling small intestinal effluent is challenging. Stool samples may provide a window into the more proximal microbial community. TYPE OF STUDY: Diagnostic. LEVEL OF EVIDENCE: Level II.

Transcriptional profiling identifies caspase-1 as a T cell-intrinsic regulator of Th17 differentiation.

Dendritic cells (DCs) are critical for the differentiation of pathogen-specific CD4 T cells. However, to what extent innate cues from DCs dictate transcriptional changes in T cells remains elusive. Here, we used DCs stimulated with specific pathogens to prime CD4 T cells in vitro and found that these T cells express unique transcriptional profiles dictated by the nature of the priming pathogen. More specifically, the transcriptome of in vitro C. rodentium-primed Th17 cells resembled that of Th17 cells primed following infection in vivo but was remarkably distinct from cytokine-polarized Th17 cells. We identified caspase-1 as a unique gene up-regulated only in pathogen-primed Th17 cells and discovered a critical role for T cell-intrinsic caspase-1, independent of inflammasome, in optimal priming of Th17 responses. T cells lacking caspase-1 failed to induce colitis or confer protection against C. rodentium infection due to suboptimal Th17 cell differentiation in vivo. This study underlines the importance of DC-mediated priming in identifying novel regulators of T cell differentiation.

Reduced anti-inflammatory gut microbiota are associated with depression and anhedonia.

INTRODUCTION: Characterise gut microbiota distributions of participants with co-occurring depression and anxiety, in those with only depression or with anxiety, and determine if gut bacteria differentially correlates with distinct clinical presentations. METHODS: Participants (10 healthy controls [mean age: 33, 60% female] and 60 psychiatric subjects; major depressive disorder (comorbid with anxiety), n = 38 [mean age: 39.2, 82% female], anxiety only, n = 8 [mean age: 40.0, 100% female], depression only without anxiety, n = 14 [mean age: 41.9, 79% female]) were characterized by psychiatric assessments. Quantitative PCR and 16S rRNA sequencing were used to characterize the gut microbiota in stool samples. RESULTS: Altered microbiota correlated with pre-defined clinical presentation, with Bacteroides (p = 0.011) and the Clostridium leptum subgroup (p = 0.023) significantly different between clinical categories. Cluster analysis of the total sample using weighted UniFrac ß-diversity of the gut microbiota identified two different clusters defined by differences in bacterial distribution. Cluster 2 had higher Bacteroides (p = 0.006), and much reduced presence of Clostridales (p<0.001) compared to Cluster 1. Bifidobacterium (p = 0.0173) was also reduced in Cluster 2 compared to Cluster 1. When evaluated for clinical charateristics, anhedonia scores in Cluster 2 were higher than in Cluster 1. LIMITATIONS: The sample is smaller and predominately female. CONCLUSIONS: Reduced or absent Clostridia was consistently seen in those with depression, independent of the presence of anxiety. Conversely, reduced Bacteroides may be more associated with the presence of anxiety, independent of the presence of depression. These differences suggest that gut microbiota distribution could help clarify the underlying pathology of comorbid clinical presentation.

Severe Gut Microbiota Dysbiosis Is Associated With Poor Growth in Patients With Short Bowel Syndrome.

BACKGROUND: Children with short bowel syndrome (SBS) can vary significantly in their growth trajectory. Recent data have shown that children with SBS possess a unique gut microbiota signature compared with healthy controls. We hypothesized that children with SBS and poor growth would exhibit more severe gut microbiota dysbiosis compared with those with SBS who are growing adequately, despite similar intestinal anatomy. MATERIALS AND METHODS: Stool samples were collected from children with SBS (n = 8) and healthy controls (n = 3) over 3 months. Gut microbiota populations (16S ribosomal RNA sequencing and metagenomic shotgun sequencing) were compared, including a more in-depth analysis of SBS children exhibiting poor and good growth. Statistical analysis was performed using Mann-Whitney, Kruskal-Wallis, and χ2 tests as appropriate. RESULTS: Children with SBS had a significant deficiency of the commensal Firmicutes order Clostridiales ( P = .025, Kruskal-Wallis) compared with healthy children. Furthermore, children with SBS and poor growth were deficient in beneficial bacteria known to produce short-chain fatty acids and had expansion of proinflammatory Enterobacteriaceae ( P = .038, Kruskal-Wallis) compared with children with SBS who were growing adequately. Using metabolic function analyses, SBS/poor growth microbiomes were deficient in genes needed for gluconeogenesis but enriched in branched and aromatic amino acid synthesis and citrate cycle pathway genes. CONCLUSIONS: Patients with SBS, particularly those with suboptimal growth, have a marked gut dysbiosis characterized by a paucity of beneficial commensal anaerobes, resulting in a deficiency of key metabolic enzymes found in the gut microbiomes of healthy children.

Metagenomic Shotgun Sequencing and Unbiased Metabolomic Profiling Identify Specific Human Gut Microbiota and Metabolites Associated with Immune Checkpoint Therapy Efficacy in Melanoma Patients.

This is the first prospective study of the effects of human gut microbiota and metabolites on immune checkpoint inhibitor (ICT) response in metastatic melanoma patients. Whereas many melanoma patients exhibit profound response to ICT, there are fewer options for patients failing ICT-particularly with BRAF-wild-type disease. In preclinical studies, specific gut microbiota promotes regression of melanoma in mice. We therefore conducted a study of the effects of pretreatment gut microbiota and metabolites on ICT Response Evaluation Criteria in Solid Tumors response in 39 metastatic melanoma patients treated with ipilimumab, nivolumab, ipilimumab plus nivolumab (IN), or pembrolizumab (P). IN yielded 67% responses and 8% stable disease; P achieved 23% responses and 23% stable disease. ICT responders for all types of therapies were enriched for Bacteroides caccae. Among IN responders, the gut microbiome was enriched for Faecalibacterium prausnitzii, Bacteroides thetaiotamicron, and Holdemania filiformis. Among P responders, the microbiome was enriched for Dorea formicogenerans. Unbiased shotgun metabolomics revealed high levels of anacardic acid in ICT responders. Based on these pilot studies, both additional confirmatory clinical studies and preclinical testing of these bacterial species and metabolites are warranted to confirm their ICT enhancing activity.

DNA screening reveals pink bollworm resistance to Bt cotton remains rare after a decade of exposure.

Transgenic crops producing toxins from the bacterium Bacillus thuringiensis (Bt) kill insect pests and can reduce reliance on insecticide sprays. Although Bt cotton (Gossypium hirsutum L.) and Bt corn (Zea mays L.) covered 26 million ha worldwide in 2005, their success could be cut short by evolution of pest resistance. Monitoring the early phases of pest resistance to Bt crops is crucial, but it has been extremely difficult because bioassays usually cannot detect heterozygotes harboring one allele for resistance. We report here monitoring of resistance to Bt cotton with DNA-based screening, which detects single resistance alleles in heterozygotes. We used polymerase chain reaction primers that specifically amplify three mutant alleles of a cadherin gene linked with resistance to Bt cotton in pink bollworm, Pectinophora gossypiella (Saunders), a major pest. We screened DNA of 5,571 insects derived from 59 cotton fields in Arizona, California, and Texas during 2001-2005. No resistance alleles were detected despite a decade of exposure to Bt cotton. In conjunction with data from bioassays and field efficacy tests, the results reported here contradict predictions of rapid pest resistance to Bt crops.

Activation of HIF-1α and LL-37 by commensal bacteria inhibits Candida albicans colonization.

Candida albicans colonization is required for invasive disease. Unlike humans, adult mice with mature intact gut microbiota are resistant to C. albicans gastrointestinal (GI) colonization, but the factors that promote C. albicans colonization resistance are unknown. Here we demonstrate that commensal anaerobic bacteria-specifically clostridial Firmicutes (clusters IV and XIVa) and Bacteroidetes-are critical for maintaining C. albicans colonization resistance in mice. Using Bacteroides thetaiotamicron as a model organism, we find that hypoxia-inducible factor-1α (HIF-1α), a transcription factor important for activating innate immune effectors, and the antimicrobial peptide LL-37 (CRAMP in mice) are key determinants of C. albicans colonization resistance. Although antibiotic treatment enables C. albicans colonization, pharmacologic activation of colonic Hif1a induces CRAMP expression and results in a significant reduction of C. albicans GI colonization and a 50% decrease in mortality from invasive disease. In the setting of antibiotics, Hif1a and Camp (which encodes CRAMP) are required for B. thetaiotamicron-induced protection against C. albicans colonization of the gut. Thus, modulating C. albicans GI colonization by activation of gut mucosal immune effectors may represent a novel therapeutic approach for preventing invasive fungal disease in humans.

BMP signals promote proepicardial protrusion necessary for recruitment of coronary vessel and epicardial progenitors to the heart.

The coronary vessels and epicardium arise from an extracardiac rudiment called the proepicardium. Failed fusion of the proepicardium to the heart results in severe coronary and heart defects. However, it is unknown how the proepicardium protrudes toward and attaches to the looping heart tube. Here, we show that ectopic expression of BMP ligands in the embryonic myocardium can cause proepicardial cells to target aberrant regions of the heart. Additionally, misexpression of a BMP antagonist, Noggin, suppresses proepicardium protrusion and contact with the heart. Finally, proepicardium explant preferentially expands toward a cocultured heart segment. This preference can be mimicked by BMP2/4 and suppressed by Noggin. These results support a model in which myocardium-derived BMP signals regulate the entry of coronary progenitors to the specific site of the heart by directing their morphogenetic movement.

Morphogenesis and cytodifferentiation of the avian retinal pigmented epithelium require downregulation of Group B1 Sox genes.

The optic vesicle is a multipotential primordium of the retina, which becomes subdivided into the neural retina and retinal pigmented epithelium domains. Although the roles of several paracrine factors in patterning the optic vesicle have been studied extensively, little is known about cell-autonomous mechanisms that regulate coordinated cell morphogenesis and cytodifferentiation of the retinal pigmented epithelium. Here we demonstrate that members of the SoxB1 gene family, Sox1, Sox2 and Sox3, are all downregulated in the presumptive retinal pigmented epithelium. Constitutive maintenance of SoxB1 expression in the presumptive retinal pigmented epithelium both in vivo and in vitro resulted in the absence of cuboidal morphology and pigmentation, and in concomitant induction of neural differentiation markers. We also demonstrate that exogenous Fgf4 inhibits downregulation all SoxB1 family members in the presumptive retinal pigment epithelium. These results suggest that retinal pigment epithelium morphogenesis and cytodifferentiation requires SoxB1 downregulation, which depends on the absence of exposure to an FGF-like signal.