Hox transcription factors influence motoneuron identity through the integrated actions of both homeodomain and non-homeodomain regions.

BACKGROUND: Hox transcription factors play a critical role in the specification of motoneuron subtypes within the spinal cord. Our previous work showed that two orthologous members of this family, Hoxd10 and Hoxd11, exert opposing effects on motoneuron development in the lumbosacral (LS) spinal cord of the embryonic chick: Hoxd10 promotes the development of lateral motoneuron subtypes that project to dorsal limb muscles, while Hoxd11 represses the development of lateral subtypes in favor of medial subtypes that innervate ventral limb muscles and axial muscles. The striking degree of homology between the DNA-binding homeodomains of Hoxd10 and Hoxd11 suggested that non-homeodomain regions mediate their divergent effects. In the present study, we investigate the relative contributions of homeodomain and non-homeodomain regions of Hoxd10 and Hoxd11 to motoneuron specification. RESULTS: Using in ovo electroporation to express chimeric and mutant constructs in LS motoneurons, we find that both the homeodomain and non-homeodomain regions of Hoxd10 are necessary to specify lateral motoneurons. In contrast, non-homeodomain regions of Hoxd11 are sufficient to repress lateral motoneuron fates in favor of medial fates. CONCLUSIONS: Together, our data demonstrate that even closely related Hox orthologues rely on distinct combinations of homeodomain-dependent and -independent mechanisms to specify motoneuron identity.

Intrinsic properties guide proximal abducens and oculomotor nerve outgrowth in avian embryos.

Proper movement of the vertebrate eye requires the formation of precisely patterned axonal connections linking cranial somatic motoneurons, located at defined positions in the ventral midbrain and hindbrain, with extraocular muscles. The aim of this research was to assess the relative contributions of intrinsic, population-specific properties and extrinsic, outgrowth site-specific cues during the early stages of abducens and oculomotor nerve development in avian embryos. This was accomplished by surgically transposing midbrain and caudal hindbrain segments, which had been pre-labeled by electroporation with an EGFP construct. Graft-derived EGFP+ oculomotor axons entering a hindbrain microenvironment often mimicked an abducens initial pathway and coursed cranially. Similarly, some EGFP+ abducens axons entering a midbrain microenvironment mimicked an oculomotor initial pathway and coursed ventrally. Many but not all of these axons subsequently projected to extraocular muscles that they would not normally innervate. Strikingly, EGFP+ axons also took initial paths atypical for their new location. Upon exiting from a hindbrain position, most EGFP+ oculomotor axons actually coursed ventrally and joined host branchiomotor nerves, whose neurons share molecular features with oculomotor neurons. Similarly, upon exiting from a midbrain position, some EGFP+ abducens axons turned caudally, elongated parallel to the brainstem, and contacted the lateral rectus muscle, their originally correct target. These data reveal an interplay between intrinsic properties that are unique to oculomotor and abducens populations and shared ability to recognize and respond to extrinsic directional cues. The former play a prominent role in initial pathway choices, whereas the latter appear more instructive during subsequent directional choices.

Implementation of a longitudinal mentored scholarly project: an approach at two medical schools.

An increasing number of medical schools have implemented or are considering implementing scholarly activity programs as part of their undergraduate medical curricula. The goal of these programs is to foster students' analytical skills, enhance their self-directed learning and their oral and written communication skills, and ultimately to train better physicians. In this article, the authors describe the approach to implementing scholarly activities at a school that requires this activity and at a school where it is elective. Both programs have dealt with significant challenges including orienting students to a complex activity that is fundamentally different than traditional medical school courses and clerkships, helping both students and their mentors understand how to "stay on track" and complete work, especially during the third and fourth years, and educating students and mentors about the responsible conduct of research, especially involving human participants. Both schools have found the implementation process to be evolutionary, requiring experience before faculty could significantly improve processes. A required scholarly activity has highlighted the need for information technology (IT) support, including Web-based document storage and student updates, as well as automatic e-mails alerting supervisory individuals to student activity. Directors of the elective program have found difficulty with both ensuring uniform outcomes across different areas of study and leadership changes in a process that has been largely student-driven. Both programs have found that teamwork, regular meetings, and close communication have helped with implementation. Schools considering the establishment of a scholarly activity should consider these factors when designing programs.

Restricted patterns of Hoxd10 and Hoxd11 set segmental differences in motoneuron subtype complement in the lumbosacral spinal cord.

During normal vertebrate development, Hoxd10 and Hoxd11 are expressed by differentiating motoneurons in restricted patterns along the rostrocaudal axis of the lumbosacral (LS) spinal cord. To assess the roles of these genes in the attainment of motoneuron subtypes characteristic of LS subdomains, we examined subtype complement after overexpression of Hoxd10 or Hoxd11 in the embryonic chick LS cord and in a Hoxd10 loss-of-function mouse embryo. Data presented here provide evidence that Hoxd10 defines the position of the lateral motor column (LMC) as a whole and, in rostral LS segments, specifically promotes the development of motoneurons of the lateral subdivision of the lateral motor column (LMCl). In contrast, Hoxd11 appears to impart a caudal and medial LMC (LMCm) identity to some motoneurons and molecular profiles suggestive of a suppression of LMC development in others. We also provide evidence that Hoxd11 suppresses the expression of Hoxd10 and the retinoic acid synthetic enzyme, retinaldehyde dehydrogenase 2 (RALDH2). In a normal chick embryo, Hoxd10 and RALDH2 are expressed throughout the LS region at early stages of motoneuron differentiation but their levels decline in Hoxd11-expressing caudal LS segments that ultimately contain few LMCl motoneurons. We hypothesize that one of the roles played by Hoxd11 is to modulate Hoxd10 and local retinoic acid levels and thus, perhaps define the caudal boundaries of the LMC and its subtype complement.

Ectopic expression of Hoxd10 in thoracic spinal segments induces motoneurons with a lumbosacral molecular profile and axon projections to the limb.

Hox genes encode anterior-posterior identity during central nervous system development. Few studies have examined Hox gene function at lumbosacral (LS) levels of the spinal cord, where there is extensive information on normal development. Hoxd10 is expressed at high levels in the embryonic LS cord but not the thoracic cord. To test the hypothesis that restricted expression of Hoxd10 contributes to the attainment of an LS identity, and specifically an LS motoneuron identity, Hoxd10 was ectopically expressed in thoracic segments in chick embryos by means of in ovo electroporation. Regional motoneuron identity was assessed after the normal period of motoneuron differentiation. Subsets of motoneurons in transfected thoracic segments developed a molecular profile normally shown by LS motoneurons, including Lim 1 and RALDH2 expression. In addition, motoneurons in posterior thoracic segments showed novel axon projections to two muscles in the anterodorsal limb, the sartorius and anterior iliotibialis muscles. At thoracic levels, we also found a decrease in motoneuron numbers and a reduction in gonad size. These last findings suggest that early and high levels of Hox expression impeded motoneuron development and neural-mesodermal interactions. Despite these adverse effects, our data indicate that Hoxd10 expression is sufficient to induce LS motoneuron identity and axon trajectories characteristic of motoneurons in the LS region.

Programming neural Hoxd10: in vivo evidence that early node-associated signals predominate over paraxial mesoderm signals at posterior spinal levels.

Studies of the programming of Hox patterns at anterior spinal levels suggest that these events are accomplished through an integration of Hensen's node-derived and paraxial mesoderm signaling. We have used in vivo tissue manipulation in the avian embryo to examine the respective roles of node- derived and other local signals in the programming of a Hox pattern at posterior spinal levels. Hoxd10 is highly expressed in the lumbosacral (LS) spinal cord and adjacent paraxial mesoderm. At stages of LS neural tube formation (stages 12-14), the tailbud contains the remnants of Hensen's node and the primitive streak. Hoxd10 expression was analyzed after transposition of LS neural segments with and without the tailbud, after isolation of normally positioned LS segments from the stage 13 tailbud, and after axial displacement of posterior paraxial mesoderm. Data suggest that inductive signals from the tailbud are primarily responsible for the programming of Hoxd10 at neural plate and the earliest neural tube stages. After these stages, the LS neural tube appears to differ from more anterior neural segments in its lack of dependence on Hox-inductive signals from local tissues, including paraxial mesoderm. Our data also suggest that a graded system of repressive signals for posterior Hox genes is present at cervical and thoracic levels and likely to originate from paraxial mesoderm.