Regulatory landscape of the Hox transcriptome

Precise regulation of Hox gene activity is essential to achieve proper control of animal embryonic development and to avoid generation of a variety of malignancies. This is a multilayered process, including complex polycistronic transcription, RNA processing, microRNA repression, long noncoding RNA regulation and sequence-specific translational control, acting together to achieve robust quantitative and qualitative Hox protein output. For many such mechanisms, the Hox cluster gene network has turned out to serve as a paradigmatic model for their study. In this review, we discuss current knowledge of how the different layers of post-transcriptional regulation and the production of a variety of noncoding RNA species control Hox output, and how this shapes formation of developmental systems that are reproducibly patterned by complex Hox networks.Fundação para a Ciência e a Tecnologia grants: ( PTDC/BEX-BID/0899/2014, SFRH/BD/51876/2012); Santa Casa da Misericordia de Lisboa grant: (SCML-MC-60-2014);info:eu-repo/semantics/publishedVersio

The function of Twist1 in the Cranial Mesoderm

The transcription factor Twist1 is a key regulator of craniofacial development. Deletion of Twist1 in the mouse embryo leads to neural tube defects, abnormal head development and mid-gestational lethality. To dissect the function of Twist1 in the cranial mesoderm (CM) beyond mid-gestation, the Mesp1-Cre transgenic line was used to delete Twist1 in the anterior mesoderm, including the progenitors of the CM. Loss of Twist1 in CM cells resulted in loss and malformations of the cranial mesoderm-derived skeleton and failure to fully segregate the mesoderm and the neural crest cells. The development of extraocular muscles was compromised whereas the differentiation of branchial arch muscles was not affected, indicating a differential requirement for Twist1 in these two types of craniofacial muscle. Surprisingly, loss of Twist1 led to the inability of the mesodermal cells to maintain their mesenchymal characteristics followed by acquisition of an epithelial-like morphology. Microarray analysis of the Twist1 deficient embryos revealed gene expression changes relating to cell–matrix interaction, blood vessel morphogenesis and regulation of Epithelial to Mesenchymal Transition (EMT). Combining the microarray data set with ChIP-sequencing identified Prrx1 and Ddr2at least two Twist1 transcriptional targets Prrx1 and Ddr2, both involved in EMT and the propagation of the mesenchymal cell characteristics. The findings presented in this thesis point to a central role of role of Twist1 in maintaining the mesenchymal architecture and the progenitor state of the mesoderm necessary for proper craniofacial development

Wnt signaling promotes AChR aggregation at the neuromuscular synapse in collaboration with agrin

Wnt proteins regulate the formation of central synapses by stimulating synaptic assembly, but their role at the vertebrate neuromuscular junction (NMJ) is unclear. Wnt3 is expressed by lateral motoneurons of the spinal cord during the period of motoneuron-muscle innervation. Using gain- and loss-of-function studies in the chick wing, we demonstrate that Wnt signaling is necessary for the formation of acetylcholine receptor (AChR) clusters without affecting muscle growth. Similarly, diaphragms from Dishevelled-1 mutant mice with deficiency in Wnt signaling exhibit defects in cluster distribution. In cultured myotubes, Wnt3 increases the number and size of AChR clusters induced by agrin, a nerve-derived signal critical for NMJ development. Wnt3 does not signal through the canonical Wnt pathway to induce cluster formation. Instead, Wnt3 induces the rapid formation of unstable AChR micro-clusters through activation of Rac1, which aggregate into large clusters only in the presence of agrin. Our data reveal a role for Wnts in post-synaptic assembly at the vertebrate NMJ by enhancing agrin function through Rac1 activation

Timed Deletion of <i>Twist1</i> in the Limb Bud Reveals Age-Specific Impacts on Autopod and Zeugopod Patterning

<div><p><i>Twist1</i> encodes a transcription factor that plays a vital role in limb development. We have used a tamoxifen-inducible Cre transgene, <i>Ubc</i>-CreERT2, to generate time-specific deletions of <i>Twist1</i> by inducing Cre activity in mouse embryos at different ages from embryonic (E) day 9.5 onwards. A novel forelimb phenotype of supernumerary pre-axial digits and enlargement or partial duplication of the distal radius was observed when Cre activity was induced at E9.5. Gene expression analysis revealed significant upregulation of <i>Hoxd10</i>, <i>Hoxd11</i> and <i>Grem1</i> in the anterior half of the forelimb bud at E11.5. There is also localized upregulation of <i>Ptch1</i>, <i>Hand2</i> and <i>Hoxd13</i> at the site of ectopic digit formation, indicating a posterior molecular identity for the supernumerary digits. The specific skeletal phenotypes, which include duplication of digits and distal zeugopods but no overt posteriorization, differ from those of other <i>Twist1</i> conditional knockout mutants. This outcome may be attributed to the deferment of <i>Twist1</i> ablation to a later time frame of limb morphogenesis, which leads to the ectopic activation of posterior genes in the anterior tissues after the establishment of anterior-posterior anatomical identities in the forelimb bud.</p></div

Quantitative RT-PCR analysis of gene expression in forelimb buds.

<p>Pairs of forelimb buds dissected from control (fl/wt) or conditional knockout (cko) E11.5 embryos harvested from mothers injected with tamoxifen at E9.5 were dissected into anterior (A) and posterior (P) halves and qRT-PCR was performed for <i>Hoxd10</i> (A), <i>Hoxd11</i> (B), <i>Hoxd13</i> (C), <i>Hand2</i> (D), <i>Grem1</i> (E) and <i>Ptch1</i> (F). Asterisk indicates p<0.05 in two-tailed t-test between anterior halves of fl/wt and cko limb buds. Reference gene was <i>Polr2a</i>. n = 4 embryos for each tissue and genotype.</p

Efficient ablation of <i>Twist1</i> expression in conditional knockout embryos.

<p>(A) qRT-PCR analysis of RNA from anterior (A) and posterior (P) halves of forelimb buds collected E11.5 from mothers injected with tamoxifen at E9.5. n = 4 pairs of forelimb buds per genotype. (B–E, B′–E′.) Immunostaining with anti-Twist1 monoclonal antibody (green) of cryosectioned wild type (fl/wt) (B, C, B′, C′) and conditional knockout (CKO) (D, E, D′, E′) E11.5 forelimb buds of embryos harvested from mothers injected with tamoxifen at E10.5. (C, C′, E, E′) Merged images showing counterstaining with DAPI (blue). (B′–E′) Higher magnification images of the boxed regions in B–E.</p

Limb skeletal phenotypes of timed conditional knockout embryos at E17.5.

<p>(A–E) forelimbs, (F–J) hindlimbs. (A, F) Wild-type, (B–E, G–J) conditional knockout embryos harvested from mothers injected with tamoxifen at E9.5 (B, G), E10.5 (C, H), E11.5 (D, I) or E12.5 (E, J). Asterisk in B indicates additional cartilage attached to radius; arrowhead indicates supernumerary digits, arrows in (A–C) marks the deltoid tuberosity, which is absent in (B). Abbreviations: c, clavicle; fe, femur; fi, fibula; h, humerus; il, ilium; is, ischium; r, radius; t, tibia; u, ulna.</p

Whole mount in situ hybridization of the forelimb bud. Expression of <i>Ptch1</i> (A, B, G, H), <i>Hand2</i> (C, D, I, J) and <i>Hoxd13</i> (E, F, K, L) in wild-type (fl/wt) or heterozygous (fl/del) embryos (A, C, E, G, I, K) were compared to CKO embryos (B, D, F, H, J, L) collected at E11.5 (A–F) or E10.5 (G–L).

<p>Asterisk indicates ectopic anterior expression domains that may correspond to the site of formation of the extra digits.</p