While testing the functions of deletion mutants in the Hox protein Ultrabithorax (Ubx), we found that the embryonic repression function of Ubx on Distal-less transcription in limb primordia is highly concentration dependent. The steep sigmoidal relationship between in vivo Ubx concentration and Distal-less repression is dependent on the Ubx YPWM motif. This suggests that Ubx cooperatively assembles a multi-protein repression complex on Distal-less regulatory DNA with the YPWM motif as a key protein-protein interface in this complex. Our deletion mutants also provide evidence for a transcriptional activation domain in the N-terminal 19 amino acids of Ubx. This proposed activation domain contains a variant of the SSYF motif that is found at the N termini of many Hox proteins, and is conserved in the activation domain of another Hox protein, Sex combs reduced. These results suggest that the N-terminal region containing the SSYF motif has been conserved in many Hox proteins for its role in transcriptional activation.

An analysis is presented of what master’s students perceive as the most challenging aspects of primary literature before and after instruction in critical reading of scientific articles. After instruction, students increasingly focus on challenges requiring critical thinking.

Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither, Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homologue, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, we demonstrate that, during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, we show that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. We therefore suggest that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as at midgastrulation.

Structured analysis of primary literature, including one flawed and two conflicting papers, highly increased students’ self-efficacy, but a significant increase in performance was observed only for some aspects of science-process skills.

Development and differentiation of the vertebrate caudal midbrain and anterior hindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. We show that Otx2 and Gbx2 perform different roles in this process. We find that ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, we propose that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient.

Gbx2 homeobox genes are important for formation and function of the midbrain/hindbrain boundary, namely the isthmic organizer. Two Gbx2 genes were identified in Xenopus laevis, differing in 13 amino acids, including a change in the homeodomain. Xgbx2a is activated earlier during gastrulation and reaches higher levels of expression while Xgbx2b is expressed later, at lower levels and has an additional domain in the ventral blood islands. Their overexpression results in microcephalic embryos with shortened axes and defects in brain and notochord formation. Both genes encode functionally homologous proteins, which differ primarily in their temporal and spatial expression patterns. ß

In undergraduate biology laboratory courses, laboratory reports can be a useful tool for teaching scientific writing, integration of source material, and information literacy; however, these teaching objectives are at times undermined by students' plagiarism. Laboratory instructors often use similarity‐matching software to detect plagiarism in laboratory reports, yet similarity hits detected with such software remain poorly characterized. In the upper division molecular biology laboratory course described here, Turnitin® routinely detected dozens of similarity hits in laboratory reports. To determine whether this abundance of similarity hits was indicative of widespread plagiarism, we analyzed similarity hits detected in 255 laboratory reports written by 135 students. Only a small minority of Turnitin® similarity matches were problematic, but over half of the laboratory reports contained at least one problem with incorporation of scientific sources (e.g., laboratory manual and scientific articles). We identified four common types of such writing problems: patchwriting, technical parroting, copying, and falsification of sources. In 18% of the laboratory reports, we detected an alarmingly superficial use of primary literature. Most of the source incorporation problems did not rise to the level of plagiarism. As a result of this study, we recommend changes in scientific writing instruction and a transition to laboratories providing more authentic research experiences. © 2019 International Union of Biochemistry and Molecular Biology, 47(4):370–379, 2019.

Long non-coding RNAs (lncRNAs) have been implicated in a variety of processes in development, differentiation, and disease. In Drosophila melanogaster, the bithorax Hox cluster (BX-C) contains three Hox genes (Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B)), along with a number of lncRNAs, most with unknown functions. Here, we investigated the function of a long non-coding RNA, lncRNA: PS4 that originates in the second intron of Ubx and is transcribed in the antisense orientation to Ubx. The expression pattern of lncRNA: PS4 is complementary to Ubx in the thoracic primordia, and the lncRNA: PS4 coding region overlaps the location of the large insertion that causes the dominant homeotic mutation Contrabithorax-1 (UbxCbx-1), which partially transforms Drosophila wings into halteres via ectopic activation of Ubx. This led us to investigate the potential role of this lncRNA in regulation of Ubx expression. The UbxCbx-1 mutation dramatically changes the pattern of lncRNA: PS4, eliminating the expression of most lncRNA: PS4 sequences from parasegment 4 (where Ubx protein is normally absent) and ectopically activating lncRNA: PS4 at high levels in the abdomen (where Ubx is normally expressed). These changes, however, did not lead to changes in the Ubx embryonic transcription pattern. Targeted deletion of the two promoters of lncRNA: PS4 did not result in the change of Ubx expression in the embryos. In the genetic background of a UbxCbx-1 mutation, the lncRNA: PS4 mutation does slightly enhance the ectopic activation of Ubx protein expression in wing discs and also slightly enhances the wing phenotype seen in UbxCbx-1 heterozygotes.