In situ mutational screening and CRISPR interference define apterous cis-regulatory inputs during compartment boundary formation

The establishment of tissue axes is fundamental during embryonic development. In the Drosophila wing, the anterior/posterior (AP) and the dorsal/ventral (DV) compartment boundaries provide the basic coordinates around which the tissue develops. These boundaries arise as a result of two lineage decisions, the acquisition of posterior fate by the selector gene engrailed (en) and of dorsal fate by the selector gene apterous (ap). While en expression domain is set up during embryogenesis, ap expression only starts during early wing development. Thus, the correct establishment of ap expression pattern with respect to en must be tightly controlled. Here we have functionally investigated the transcriptional inputs integrated by the “early” ap enhancer (apE) and their requirement for correct boundary positioning. Detailed mutational analyses using CRISPR/Cas revealed a role of apE in positioning the DV boundary with respect to the AP boundary, with apE mutants often displaying mirror-image anterior wing duplications. We then accomplished tissue-specific enhancer disruption via dCas9 expression. This approach allowed us to dissect the spatio-temporal requirement of apE function, challenging the mechanism by which apE miss-regulation leads to AP defects. Base-pair resolution analyses of apE uncovered a single HOX binding site essential for wing development, which, when mutated, led to wingless flies. Along these lines, we found that the HOX gene Antennapedia (Antp) is fundamental for ap expression. In addition, we demonstrated that the transcription factors Pointed (Pnt), Homothorax (Hth) and Grain (Grn) are necessary for apE function. Together, our results provide a comprehensive molecular basis of early ap activation and the developmental consequences of its miss-regulation, shedding light on how compartmental boundaries are be set up during development.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

Induction and Dorsoventral Patterning of the Telencephalon

Here we reply to the “Refutation” of Lawrence, Casal, de Cellis, and Morata, who critique our paper presenting evidence for an organizer and compartment boundary subdividing the widely recognized posterior wing compartment of butterflies and moths (Lepidoptera) and Drosophila, that we called the F-P boundary. Lawrence et al. present no data from the Lepidoptera and while the data that they present from Drosophila melanogaster mitotic clones are intriguing and may be informative with respect to the timing of the activity of the A-P and F-P organizers, considerable ambiguity remains regarding how their data should be interpreted with respect to the proposed wing compartment boundaries. Thus, contrary to their claims, Lawrence et al. have failed to falsify the F-P boundary hypothesis. Additional studies employing mitotic clones labeled with easily detectable markers that do not affect cytoskeletal organization or rates of cell division such as GFP and RFP clones produced by G-Trace or Twin Spot Generator (TSG) may further clarify the number of compartment boundaries in Drosophila wings. At the same time, because Drosophila wings are diminutive and highly modified compared to other insects, we also urge great caution in making generalizations about insect wing development based exclusively on studies in Drosophila.Replying to: Lawrence, P.A., Casal, J., de Celis, J., Morata, G. A refutation to ‘A new A-P compartment boundary and organizer in holometabolous insect wings’. Sci. Rep. 9 (2019), https://doi.org/10.1038/s41598-019-42668-y.

A P element enhancer trap screen was conducted to identify genes involved in dorsal-ventral boundary formation in Drosophila. The son of Notch (son) gene was identified by the son(2205) enhancer trap insertion, which is a partial loss-of-function mutation. Based on son(2205) mutant phenotypes and genetic interactions with Notch and wingless mutations, we conclude that son participates in wing development, and functions in the Notch signaling pathway at the dorsal-ventral boundary in the wing. Notch signaling pathway components activate son enhancer trap expression in wing cells. son enhancer trap expression is regulated positively by wingless, and negatively by cut in boundary cells. Ectopic Son protein induces wingless and cut expression in wing discs. We hypothesize that there is positive feedback regulation of son by wingless, and negative regulation by cut at the dorsal-ventral boundary during wing development.

Unraveling the inner workings of neural circuits entails understanding the cellular origin and axonal pathfinding of various neuronal groups during development. In the embryonic hindbrain, different subtypes of dorsal interneurons (dINs) evolve along the dorsal-ventral (DV) axis of rhombomeres and are imperative for the assembly of central brainstem circuits. dINs are divided into two classes, class A and class B, each containing four neuronal subgroups (dA1-4 and dB1-4) that are born in well-defined DV positions. While all interneurons belonging to class A express the transcription factor Olig3 and become excitatory, all class B interneurons express the transcription factor Lbx1 but are diverse in their excitatory or inhibitory fate. Moreover, within every class, each interneuron subtype displays its own specification genes and axonal projection patterns which are required to govern the stage-by-stage assembly of their connectivity toward their target sites. Remarkably, despite the similar genetic landmark of each dINs subgroup along the anterior-posterior (AP) axis of the hindbrain, genetic fate maps of some dA/dB neuronal subtypes uncovered their contribution to different nuclei centers in relation to their rhombomeric origin. Thus, DV and AP positional information has to be orchestrated in each dA/dB subpopulation to form distinct neuronal circuits in the hindbrain. Over the span of several decades, different axonal routes have been well-documented to dynamically emerge and grow throughout the hindbrain DV and AP positions. Yet, the genetic link between these distinct axonal bundles and their neuronal origin is not fully clear. In this study, we reviewed the available data regarding the association between the specification of early-born dorsal interneuron subpopulations in the hindbrain and their axonal circuitry development and fate, as well as the present existing knowledge on molecular effectors underlying the process of axonal growth.

The positional cues for formation of individual inner ear components are dependent on pre-established axial information conferred by inductive signals from tissues surrounding the developing inner ear. This review summarizes some of the known molecular pathways involved in establishing the three axes of the inner ear, anterior-posterior (AP), dorsal-ventral (DV) and medial-lateral (ML). Signals required to establish the AP axis of the inner ear are not known, but they do not appear to be derived from the hindbrain. In contrast, the hindbrain is essential for establishing the DV axis of the inner ear by providing inductive signals such as Wnts and Sonic hedgehog. Signaling from the hindbrain is also required for the formation of the ML axis, whereas formation of the lateral wall of the otocyst may be a result of first establishing both the AP and DV axes. In addition, this review addresses how genes induced within the otic epithelium as a result of axial specification continue to mediate inner ear morphogenesis.

Zbtb21 is required for the anterior-posterior patterning of neural tissue in the early Xenopus embryo

Regulation of expression of Vg and establishment of the dorsoventral compartment boundary in the wing imaginal disc by Suppressor of Hairless

Wingless Modulates the Effects of Dominant Negative Notch Molecules in the Developing Wing of Drosophila

Germband extension during Drosophila development features the merging of cells along the dorsal–ventral (DV) axis and their separation along the anterior–posterior (AP) axis. This intercalation process involves planar cell polarity, anisotropic contractile forces along cell edges, and concerted cell deformation and movement. Although prior experiments have probed each of these factors separately, the connection among them remains unclear. This paper presents a chemo-mechanical model that integrates the three factors into a coherent framework. The model predicts the polarization of Rho-kinase, myosin and Bazooka downstream of an anisotropic Shroom distribution. In particular, myosin accumulates on cell edges along the DV axis, causing them to contract into a vertex. Subsequently, medial myosin in the cells anterior and posterior to the vertex helps to elongate it into a new edge parallel to the body axis. Thus, the tissue extends along the AP axis and narrows in the transverse direction through neighbor exchange. Model predictions of the polarity of the proteins and cell and tissue deformation are in good agreement with experimental observations.

Neuronal branching is essential for establishing complex neural circuits. Many axons and dendrites in the CNS branch, forming complex process arbors. Some of these branches are formed during initial outgrowth and often occur via bifurcation of the growth cones of the extending neuronal processes. However, others are formed de novo by branch extension from a previously existing neurite. This process is often referred to as collateral branching. Here, we investigate the molecular mechanisms underlying this process. We show that in C. elegans, collateral branching of the PLM neuron is mediated by an actin assembly process guided by Transducer of CDC-42-Dependent Actin assembly-1 (TOCA-1). This scaffolding protein is recruited to the branching site at the time of branch formation. cdc-42 and the guanine nucleotide exchange factor dock-11 are also required for branch formation. Biochemically, TOCA-1 has been demonstrated to recruit WASP-1 and activate the Arp2/3 complex to promote actin assembly. In vivo, both wsp-1 and the Arp2/3 complex mutants also disrupt branch formation. While loss of TOCA-1 disrupts branching, it does not influence anterior-posterior (AP) or dorsal-ventral (DV) positioning of the branch, in contrast to previously defined branching regulators. Our data support a model in which TOCA-1 acts downstream of AP and DV positioning factors, directly orchestrating filopodial extension to form the nascent branch.

The inner ear develops from an ectodermal thickening known as the otic placode into a complex structure that is asymmetrical along both the anterior-posterior (A-P) and dorsal-ventral (D-V) axes. Embryological manipulations in Xenopus allow us to test regenerative potential along specific axes and timing of axis determination. We explore the role of Wnt signaling with gain and loss of function experiments. In contrast to A or P half ablations, D or V half ablations almost never result in mirror duplications or normal ears. Instead there is a loss of structures, especially those associated with the ablated region. Rotation experiments inverting the D-V axis reveal that it is determined by stage 24-26 which is just before expression of the dorsal otic marker Wnt3a. Conditional blocking of canonical Wnt signaling results in reductions in the number of sensory organs and semicircular canals which could be placed in one of three categories, the most common phenotypes being similar to those seen after dorsal ablations. There is less regenerative potential along the D-V axis. Wnt3a protein alone is sufficient to rescue the severe loss of inner ear structures resulting from dorsal but not ventral half ablations.

There has been considerable progress in understanding how restricted patterns of gene expression and differentiation are set up along the anteriorposterior (A-P) and dorsal-ventral (D-V) axes of the vertebrate embryo, and how these provide the fundamental framework for regionalized visceral, neural and cardiovascular organogenesis. Intimately linked, and in many ways equally important, is the morphogenesis that occurs orthogonal to these axes and leads to the left-right (L-R) asymmetric body plan. This plan is evident in all vertebrates as a stereotypical looping of the heart, gut, and the positioning of associated organs. In humans, complete inversion of the normal L-R body plan, called situs inversus, is not deleterious to health, but situations in which some tissues adopt the normal orientation while others are inverted (heterotaxia or situs ambiguus), have profound medical consequences, such as improper connections of the large blood vessels and abnormally chambered hearts (see Kathiriya and Srivastava 2000). Up to 1 in 8500 of human births have congenital defects associated with laterality defects, but there are likely to be many more losses that are unaccounted for in utero. Although the L-R asymmetry program appears to be quite sensitive to a variety of environmental and genetic disturbances, the overall uniformity of L-R patterning both within and between vertebrate species implies a strong, and to some degree conserved, underlying genetic regulation.KeywordsSitus InversusNodal SignalingZebra FishPineal OrganHeart TubeThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Determination of EGFR Signaling Output by Opposing Gradients of BMP and JAK/STAT Activity