Analysis of Development Edited by Prof Benjamin H Willier, Prof Paul A Weiss and Prof Viktor Hamburger Pp xii + 735 (Philadelphia and London: W B Saunders Company, 1955) 105s

Mechanisms of Development

Robo4 is a vascular-specific receptor that inhibits endothelial migration

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a repulsive guidance system that prevents inappropriate axons from crossing the central nervous system midline; this repulsive system is mediated by the secreted extracellular matrix protein Slit and its receptors Roundabout (Robo). Three distinct slit genes (slit1, slit2, and slit3) and three distinct robo genes (robo1, robo2, rig-1) have been cloned in mammals. However, to date, only Robo1 and Robo2 have been shown to be receptors for Slits. In rodents, Slits have been shown to function as chemorepellents for several classes of axons and migrating neurons. In addition, Slit can also stimulate the formation of axonal branches by some sensory axons. To identify Slit-responsive neurons and to help analyze Slit function, we have studied, by in situ hybridization, the expression pattern of slits and their receptors robo1 and robo2, in the rat central nervous system from embryonic stages to adult age. We found that their expression patterns are very dynamic: in most regions, slit and robo are expressed in a complementary pattern, and their expression is up-regulated postnatally. Our study confirms the potential role of these molecules in axonal pathfinding and neuronal migration. However, the persistence of robo and slit expression suggests that the couple slit/robo may also have an important function in the adult brain.

Spatiotemporal expression patterns of slit and robo genes in the rat brain.

Mesenchymal stem cells (MSCs) were initially identified by their capacity to differentiate into connective tissue cell types. In the past decade MSCs were also shown to exhibit unexpected plasticity, which was thought to account for their broad therapeutic efficacy in animal models of disease and human clinical trials. More recent evidence indicates that their capacity to alter the microenvironment via secretion of soluble factors contributes more significantly than their plasticity in effecting tissue repair. However, the production by MSCs of a diverse array of trophic factors is inconsistent with their designation as stem cells, which by definition lie at the apex of a hierarchy of cellular differentiation and lineage specification. Analysis of the MSC transcriptome has led to the identification of sub populations that express a variety of regulatory proteins that function in angiogenesis, hematopoiesis, neural activities, and immunity and defense. These activities reflect the varied functions of distinct stromal subtypes in marrow that play important roles in tissue homeostasis. Evidence is provided that the biochemical heterogeneity of these subpopulations contributes more significantly to the therapeutic potential of MSCs than their stem-like characteristics.

Biochemical heterogeneity of mesenchymal stem cell populations: clues to their therapeutic efficacy.

Congenital diaphragmatic hernia, kidney agenesis and cardiac defects associated with Slit3-deficiency in mice

Expression patterns of Slit and Robo family members during vertebrate limb development.

We report the cloning and expression during limb development of the chicken Slit1, Slit2, and Slit3 ligands, and Robo1 and Robo2 receptorgenes. We also compare the expression patterns of Robo1 and Robo2 in developing chick and mouse hindlimbs. These genes are expressed inregions of muscle development, chrondrogenesis, and axon guidance. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Roundabout; Robo1; Robo2; Rig1; Slit1; Slit2; Slit3; Chick; Mouse; Limb development; Muscle; Innervation; Axon guidance; Chrondrogenesis;Gene expression; Apical ectodermal ridge; Mesenchyme 1. Results and discussionThe Slit ligands and their Robo receptors regulate axonalprojections and myoblast migration in invertebrates (Kidd etal., 1998, 1999). In vertebrates, they regulate axon guidanceand branching, and Slit family gene expression patterns indeveloping murine limbs suggest additional, non-neuralfunctions (Dickson, 1998; Holmes et al., 1998; Harris andHolt, 1999; Guthrie, 1999; Yuan et al., 1999b; Brose andTessier-Lavigne, 2000; Kaprielian et al., 2000; Tear, 2001).Neither Robo1 nor Robo2 expression has been reported inthe developing avian or murine limb. Furthermore neitherthe cloning nor detailed expression patterns of the avian Slitgenes during limb development has been reported.1.1. Cloning of the chick Slit1, Slit2, Slit3, Robo1 and Robo2genes and their expression in developing limbsWe cloned partial cDNAs of three chicken Slit and twochicken Robo genes. These genes were named Slit1, Slit2,Slit3, Robo1 and Robo2 based on amino acid sequence simi-larities to other vertebrate Slit and Robo proteins (Fig. 1).Expression patterns in developing chick limbs fromHamburger-Hamilton stage 20 through stage 32 (HH s20–s32) for each of these genes were obtained by non-radio-active whole mount and section in situ hybridization histo-chemistry (Hamburger and Hamilton, 1951; Figs. 2–6).From HH s20–s23, Slit1 is expressed in proximal limbmesenchyme (Fig. 2A), while Slit2 is observed in distalmesenchyme underlying the apical ectodermal ridge(AER; Fig. 2E). In contrast Slit3 expression is restricted tothe AER ectoderm (Fig. 2I). Robo1 is expressed in bothproximal central mesenchyme and the AER (Fig. 2M).Robo2 is expressed in proximal central and anteriormesenchyme but not the AER (Fig. 2Q). Both Slit3 andRobo1 expression in the AER is maintained through atleast HH s29.By HH s25 these expression domains are more elaborate.Slit1 and Slit2 are expressed in an irregular horseshoepattern in both dorsal and ventral mesenchyme, whileSlit3 is expressed more centrally (Figs. 2B,F,J and 3A–C).All three Slit genes are also expressed in the peripheralmesenchyme, although Slit2 expression is morepronounced, especially towards the anterior and distalmargins (Fig. 2F). Robo1 expression intensiﬁes and expandsto ﬁll the proximal central mesenchyme, and is also presentat the periphery (Figs. 2N and 3D). Robo2 expression ismaintained predominantly in proximal, central and anteriormesenchyme (Fig. 2R). These central mesenchyme expres-sion domains are similar to those of muscle markers (datanot shown; Buscher and Izpisua-Belmonte, 1999).

Izabella Messina

Papers

Expression patterns of Slit and Robo family members during vertebrate limb development.

Gene expression pattern Expression patterns of Slit and Robo family members during vertebrate limb development

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