Sst) cells from E10.5 onward, comparing their topography with the expression patterns of the genes Dlx5, Gbx2, Lhx7-8, Nkx2.1, Nkx5.1 (Hmx3), and Shh, which variously label parts of the subpallium. Whereas some experimental results suggest that Sst cells are pallidal, our data reveal that many, if not most, telencephalic Sst cells derive from de diagonal area (Dg). Sst-positive cells initially only present at the embryonic Dg selectively populate radially the medial part of the bed nucleus striae terminalis  and part of the central amygdala; they also invade tangentially the striatum, while eschewing the globus pallidum and the preoptic area, and integrate within most cortical and nuclear pallial areas between E10.5 and E16.5.The telencephalic subpallium is the source of various GABAergic interneuron cohorts that invade the pallium via tangential migration. Based on genoarchitectonic studies, the subpallium has been subdivided into four major domains: striatum, pallidum, diagonal area and preoptic area  contains supplementary material, which is available to authorized users. The identity of neuron types produced within a specific brain region is the result of progressive patterning and consequent fate specification of progenitors during early ontogeny. Establishment of a unique molecular profile at a given progenitor domain allows it to generate a particular neuronal cell type . Such neurons are presumed to be different at least in some subtle aspects from those produced in adjoining areas, irrespective that some part of the respective molecular profiles may be shared. Some neuronal derivatives aggregate radially within the local mantle zone, whereas others may migrate tangentially into neighbouring or distant brain areas. The latter is a well-known phenomenon in the telencephalon, where various subpallial cell populations migrate into other parts of the subpallium or into the pallium, contributing diverse contingents of inhibitory interneurons to local circuitry (reviewed in Marín and Rubenstein preoptic area (POA), diagonal area (Dg), pallidum , and striatum (St)  can be distinguished generically across each of these main domains, forming parallel series along the septoamygdaloid axis , as well as by Puelles et al.  Fig. . HistoriSt) Fig. . This isSt) Fig.  identifiSt) Fig. c. FindinSt) Fig. . This naxis Fig. b. These s et al. . The cens et al. . The cors et al. . At the s et al. b.Fig. 1-The molecular phenotype of the subpallial ventricular and subventricular zone was used by Flames et al.  to definMost neurons generated from these different subpallial progenitor areas are GABAergic. Some of them settle radially into the local mantle and differentiate as projection neurons or interneurons. In addition, groups of GABAergic and cholinergic neurons migrate tangentially from some subpallial areas into other subpallial areas  was first isolated from hypothalamic extracts on the basis of its ability to inhibit growth hormone secretion from the anterior pituitary  was specifically proposed as the main source of Sst-expressing cortical interneurons by Wonders et al. . The present full descriptive data accordingly provide information about the appropriateness of E12.5/E13.5 material for the deductions obtained from those transgenic experiments.According to in vitro and in vivo fate-mapping studies, the medial ganglionic eminence (MGE), which is the sum of the central parts of Pal, Dg and POA Fig. b, is curs et al. , a concls et al. . HoweverSst mRNA expression during early development in the mouse telencephalon, aiming to trace overall developmental distribution of this cell type in the telencephalon, starting at initial stages. In order to illuminate the issue of a potential localized source within a subdomain of the MGE , Sst mRNA expression was compared in adjacent sections with Shh signal and several differentially expressed transcription factors  over the embryonic period E9.5 to E16.5. Our study led us to pinpoint the Dg domain as the first subpallial domain that contains cells expressing Sst in its mantle (from E10.5 onwards). At this time point, these diagonal Sst cells clearly are topographically distinct from pallidal Gbx2-positive cells, as well as from preoptic Shh/Nkx5.1-positive derivatives. At E12.5, many Sst cells apparently derived from the Dg domain have already invaded tangentially the striatal mantle  and start to migrate subpially past the LGE into the pallium. The tangentially migrated Sst population increases markedly subsequently, but no other locus  was found outside the Dg where Sst cells clearly seem to arise from the ventricular or subventricular zone. However, our material may not be sufficient to negate altogether that possibility, since the expression of Sst may start after some delay. Our analysis accordingly suggests that numerous Sst-expressing neurons that colonize cortical and subcortical structures seem to derive tangentially from the Dg domain. Our data indicate that the Sst-positive components that eventually populate the medial bed nucleus striae terminalis complex and a part of the central amygdala represent radial derivatives of the Dg, produced along the length of its septoamygdaloid dimension.In the present work, we used the updated model of subpallial areal subdivisions Fig.  to analyAll experimental procedures involving use and care of laboratory animals were conducted in compliance with the current normative standards of the European Community (86/609/EEC) and the Spanish Government .Swiss albino mouse embryos were collected from embryonic day (E) 9.5–16.5 after fertilization (adult specimens were collected as well). Noon on the day of the appearance of the vaginal plug was considered day 0.5 of gestation (E0.5). Mouse embryos were separately staged according to the Theiler stages  cDNA fragments were obtained by reverse transcription (RT). RNA was individually extracted with Trizol reagent  from freshly dissected brains of Mus musculus embryos at E10.5, E12.5, and E14.5. The RNA was treated with DNase I  for 15 min at room temperature (RT), and the enzyme was then inactivated at 65 °C. Afterward, RNA samples were converted to single-stranded cDNA with Superscript III reverse transcriptase  and oligo-dT-anchored primers. The resulting first-strand cDNA (0.5 μl of the reverse transcription reaction) was used as a template for the PCR reaction, which was performed in presence of Taq polymerase  and the following gene-specific primers for Lhx8 and Nkx5.1 (Hmx3) mRNA.mLhx8F: 5′-AGCTGGTATGTGACGAGCA-3′mLhx8R: 5′-AGAATGGTTGGGACTGACG-3′mNkx5.1F: 5′-GACCACAAGGAGCTGGACTC-3′mNkx5.1R: 5′-TAAGAGGAGAAGCGCCTCAA-3′The PCR conditions used were an initial denaturation step at 94 °C for 5 min, then 35 cycles , followed by 20 min at 72 °C. The PCR products were cloned into the pGEM-T Easy Vector , and sequenced .Dlx5, Gbx2, Lhx8, Nkx2.1, Nkx5.1,Shh, and Sst were synthetized with a kit, following the manufacter´s recommendations , and applying specific polymerases . Plasmid information is provided in Table http://www.developingmouse.brain-map.org), as well as our own previously published studies on the subject.The embryos were processed for in situ hybridization with digoxigenin-UTP-labeled antisense riboprobes. Sense and antisense digoxigenin-labeled riboprobes for mouse Our immunohistochemical reaction protocol has been described in detail elsewhere . After scanning, the visualization and capture of images of adjacent labeled sections were carried out by using the Aperio software ImageScope. The images were corrected for contrast, focus, and brightness. In order to compare different gene expression patterns, the images of adjacent sections reacted with different markers were superposed and artificially pseudocolored (from blue to red or green) with Photoshop CS3. The plates were labeled using Adobe Photoshop Illustrator CS2 .Sst signal was detected in a restricted sector of the subpallial mantle zone from E10.5 onward , as well as in cells that migrate selectively from there into the pallidal mantle   and preoptic (POA) domains, excluding the striatum (St) .A more precise mapping of the precocious hh Figs. a–r, 5a–xShh Fig. , or Shh Gbx2 cells lie superficial to the large ventricular/subventricular zone, mainly in the rostral half of the MGE ; it limits with the Dg across the hp2/hp1 border . Overlap comparison of these patterns indicates again that the Dg Sst cells lie precisely underneath and outside the Pal Gbx2 cells , and penetrating tangentially the striatal marginal zone   becomes apparent; this forms a distinct mantle domain that extends caudalwards with progressively fewer and deeper cells, always found underneath the Dg domain populated by Sst cells  Fig. . This trx3) Fig. . At 10.5x3) Fig. . In rostrea Fig. a–d. Otherea Fig. d. Most olls Fig. a–c. In ceas Fig. i, j, k. Lhx7-8 expression in transverse sections with the studied Sst and Shh patterns. Lhx7-8 is a general marker for Pal, Dg, and POA, thus offering a contrast with the more selective Sst and Shh signals . There are also many marked cells scattered in the subventricular zone of the Pal domain, particularly at rostral section levels, where these elements are relatively more numerous in the medial part of the MGE than laterally, and the corresponding medial mantle stratum is also more massively populated . The Dg domain, as defined by the weak and patchy ventricular expression of Shh, seems to contribute likewise to this mz/vz Lhx7-8 pattern, though it shows a thinner positive mantle . At the diagonal part of the septum (median SeDg area), only a positive mantle zone was present , suggesting a possible tangential migration from more lateral origins. Once the POASe area is reached in the series of sections, the Lhx7/8+ mantle zone disappears . At a single section level, there was a marginal line of labeled cells at the POA2 (Shh-negative) area . More caudally, there appear instead Lhx7/8 cells in the POA1 mantle , which is distinctly thinner than that present at the Pal/Dg complex . The Lhx7-8+ Pal mantle population diminishes in cell density caudalwards, coinciding with the place where Sst cells course marginally through the Pal into the St . Though there is some topographic overlap between Sst and Lhx7/8 cells at the Dg domain and neighbouring Pal, many tangentially migrating Sst cells clearly do not express Lhx7/8 . It is unclear whether this implies that these cells downregulate an initial postmitotic expression of Lhx7-8.Finally, we also compared Sst cells streaming tangentially through the subpial part of the central subpallium start to invade the striatum and the pallium at E11.5 ; the latter appears disposed as an oblique  band of labeled cells at the back of the SSpM. The labeled diagonal population is sparse superficially at the site of the prospective diagonal band nuclei  and dorsolateral to the preoptic area  part of the supracapsular BST (BSTMsc), since the lateral BST counterpart belongs to the periventricular pallidum . Ventral to the level where the internal capsule penetrates the subpallium (closer to the olfactory tuberculum), the caudal end of the supracapsular BSTM arch extends into the amygdala, where we observe the dense radially migrated Sst cells of the amygdaloid BST complex (BSTA) and the associated diagonal part of the central amygdala (CA), as well as other Sst cells that migrated tangentially into the pallial amygdala, invading mainly the prospective basal and cortical region, and the amygdalo-hippocampal area, but eschewing the medial amygdala  shows its characteristic oblique band of dense SI; Fig. f, g; sucSI; Fig. h–j.Fig. Sst and Nkx2.1  illustrates at E13.5 that Sst cells are practically absent in the globus pallidus  from the Nkx2.1+/Sst+ diagonal domain (with weak Nkx2.1).At E13.5, the striatal mantle shows a dispersed population of labeled cells, whose density markedly increases near the pial surface , and reaches a maximum at the SSpM stream, which lies now deep to the incipient olfactory tuberculum  and the diagonal-septal area ; interestingly, this does not occur at the intervening pallido-septal area  , whereas the neighboring supracapsular migration stream that vehiculates hypothalamic Otp-positive neurons into the amygdala passes just medial to the BSTM, outside the Dlx5-positive subpallium, within a thin periventricular pallial corridor that connects the pallial amygdala with the peduncular hypothalamus , which leads us to propose renaming it the magnocellular diagonal nucleus, or DgMC , many of these cells form definitive nuclear derivatives, irrespective that others may enter the SSpM and continue tangentially into the cortex or the striatum. These local radial mantle derivatives of the amygdaloid part of the diagonal area correspond to the primordium of the lateral central amygdala, found next to the BSTMa , a larger triangular pallial aggregate also held to derive from the ventral pallium  a nearly mature appearance. The distribution remains gradiental along the radial dimension extending from the olfactory tuberculum, past the ventral striatum, into the main body and its subventricular zone at E15.5, , at the transition between dorsal and ventral striatum regions . The supracapsular BSTL is practically devoid of Sst cells, particularly at its subventricular zone, though some Sst cells characterize the local mantle deep to the internal capsule .Found along the radial dimension of the Pal domain extending past the GP into the subpial olfactory tubercle, the ventral pallidum  shows increased presence of Sst cells. The periventricular derivative is the BSTM, which appears largely as a thin strongly Sst-positive band in sagittal sections , another constituent of the central diagonal intermediate stratum, which displays a dispersed population of Sst cells . We believe this interpretation is consistent with corresponding data shown by García-López et al.  in the Allen Developing Mouse Brain Atlas (online since 2009) and Puelles et al. . This reThe Dg was previously named ‘anterior entopeduncular area’ in early reports on the prosomeric model  and lacks Couptf1 signal, whereas the opposite is true for the pallidum. Present data illustrate a remarkably precise correlation of Sst neurons with the mantle of this domain; García-López et al.  to have a distinctive name for the stalk area, leaving aside the old term AEP, as well as the vague descriptors of its position within the MGE. The recently updated prosomeric model ; this conveys the correct topographic linear extent along the oblique hemispheric stalk and septoamygdaloid axis, and is based on a well-known classic concept and related identifiable surface landmark, the diagonal band . This name refers to the adult region and corresponding characteristic cell populations, irrespective whether these are produced or not within the Dg proper .Curiously, Flames et al.  resolvedz et al.  previousIn summary, the narrow diagonal radial histogenetic domain defined at the hemispheric stalk Dg; Fig. d is travSst cells in the mouse telencephalon between E10.5 and E16.5. In the final distribution such cells populate within the pallium the whole cerebral cortex (a well-studied point we do not need to examine in detail), the claustrum and the pallial amygdala, showing scarce contributions to the olfactory bulb. Sst cells also invade the subpallium, though differentially. They are massively present in the lateral part of the central amygdaloid nucleus, as well as in the medial part of the bed nucleus stria terminalis complex, both of which are understood here as intrinsic  derivatives of the diagonal area  roughly corresponds to the pMGE5 ventricular progenitor domain of Flames et al. , as well as with transcripts of Shh, the gene coding the morphogen SHH, significantly expressed in a large population of preoptic cells that invade the pallidum , whereas García-López et al. , in agreement with our present conclusions. In the following sections, we will comment on the development of telencephalic Sst cells, and discuss the conflicting views about the origin of these cells.Authors who examined the origin of cortical z et al. , Real etz et al.  and Mediz et al.  associatSst cells in the developing mouse telencephalon. Given the existence of several experimental transgenic tracings demonstrating that such cells migrate tangentially from a restricted (single) subpallial source into other telencephalic domains (see citations below), we have parsimoniously interpreted the observed absolute changes in topography as evidence of tangential migration, starting at the apparent source . Our results revealed that the earliest Sst cells identified at E10.5 are not uniformly distributed within the MGE, being restricted to a thin radial domain of the MGE mantle zone that appears intercalated between the pallidal and preoptic regions, as identified by differential markers. This is precisely the estimated position of the Dg . The subpallial nature of the diagonal Sst cell population is corroborated by the shared expression of general subpallial markers such as Dlx5, Nkx2.1, and Lhx7/8.In the present report, we mapped descriptively the progressive changes observable in the topography of Sst cells is continuous superficially with an incipient subpial aggregate of similar cells spreading tangentially lateralwards; at this stage, these subpial cells partially cover marginally the pallidal mantle core occupied selectively by Gbx2- and Shh-positive cells. A few of these subpial Sst elements even penetrate the striatal marginal stratum . This superficial subpallial migration stream (abbreviated as SSpM) becomes much better developed at subsequent stages (E11.5-E14.5). It clearly represents the main pathway for the arrival of diagonal Sst cells at the striatum  and the pallium . Our data indicate that the alternative intermediate and subventricular migration pathways so common for other subpallial migrating cortical interneurons . This apparently reveals a persistent non-permissive or repellent pallidal effect on the migrating Sst cells; this phenomenon may explain as well the paucity of Sst cells that migrate subventricularly into the striatum and the cortex, since they need to cross the pallidal territory. Moreover, no Sst cells were ever observed within the preoptic area; this result indicates a clearcut spatial orientation of the SSpM in the opposite palliopetal direction, possibly influenced by repellent signals spreading out of the preoptic area. It may be speculated whether SHH highly present in the preoptic and pallidal environments is repulsive for the Sst cell population , the pallidal mantle core remains practically devoid of Sst cells that migrate tangentially away from the Dg via the SSpM and the deep and superficial amygdalar streams, the Dg histogenetic area also develops radial derivatives that mature locally; these appear thereafter clearly intercalated in the subpallial mantle between the Sst-negative pallidal and preoptic areas. This locus corresponds to a radial domain placed obliquely along the telencephalic stalk, which has received little embryological attention per se so far, but is known to exist since the nineties . The BSTM also includes some supra- and subcapsular elements classified by some authors within the medial extended amygdala  we saw rather sparse Sst cells at the level of the diagonal band nuclei, together with a rather dispersed Sst population within the innominate area deep to them; in contrast, there appears a well-developed population of large and small Sst cells centered within the periventricular BSTM formation. Importantly, the latter population was found to lie just outside the pallidal domain expressing Nkx2.1 (or NKX2.1) from E13.5 onward , while it clearly lies within the Dlx5-expressing subpallial mantle , and therefore does not coincide with the Sst-positive BSTM; it demonstrably passes just outside the Dlx5-positive domain that contains the subpallial Sst BSTM elements . These authors just assumed a local subpallial origin of the cholinergic cell type, without commenting on the potential role of diverse MGE progenitor subareas in their production, an issue subsequently underlined by Flames et al.  may bespeak of an indirect non-cell-autonomous effect due to local MGE mantle conditions controlled by Lhx8. According to the results of Pombero et al. , and further supports the notion that the Dg is a molecularly distinct from the Pal  probably represent a significant part of the o et al.  reporteds et al. . The demo et al.  such an o et al.  and Pombo et al. . The faco et al.  observedSst cells from E14.5 onward; it is first visible as an emergent aggregate of Sst cells at E13.5 . The BSTM supracapsular arch extends across the paraseptal Dg area (which neighbours the crossing fibers of the anterior commissure) into the diagonal part of the subpallial medial septum, where individual parts of the BSTM formation have been recognized in adult rodents (these are often described as ‘anterior BST’ in the relevant literature). The amygdaloid BST nucleus is wholly or in part a caudal prolongation of the supracapsular BSTM into the amygdaloid part of the subpallium . Bupesh et al. (ER81 and Six3 expression). This arrangement had not been disclosed so far.The potential existence of intrinsic amygdaloid Dg derivatives—and h et al.  reportedh et al. p, q, 12,Sst-positive population identifiable as an incipient radial migratory stream at the amygdaloid Dg subarea already at E12.5  is a compactly Sst cells invade the pallial amygdala, either via the subpial SSpM, or via a specific, well-developed, amygdaloid subventricular migratory pathway. We could not assess whether these cells originate specifically from paraseptal, central, or amygdaloid parts of the Dg area, or come from all of them. A feature suggesting a general Dg origin is that the earliest cells invading the pallial amygdala via the SSpM were observed at E11.5 , basomedial (BM) and amygdalohippocampal (AHi) amygdaloid nuclear primordia. There is the apparent exception of the basolateral, posteromedial cortical, and medial amygdaloid nuclei, which stand out as loci with few Sst neurons , a formation derived from the ventral pallium that was recently distinguished at this locus .At E16.5, the L primordium appears densely populated and even delineated by lls Fig. f–h. Hori Puelles ; this pr Puelles . The amy Puelles . There a Puelles . At postSst cells was largely centered on the chronology of the arrival of these cells to the different areas. By E16.5 the whole cortex was covered superficially by Sst cells. As mentioned above, we believe that the main pathway for this tangential migration is the SSpM. Following Puelles . As was previously reported, the major adult population of cortical Sst interneurons has a subgranular topography . Presumably, labeled embryonic cells occupying a similar marginal position at E15.5 and E16.5 will be distributed to the sparser population later found in the supragranular layers.Our analysis of the invasion of the isocortical plate by  Puelles , we inteDlx5/6-expressing subpallial lineage ; see also Puelles et al. , and including comparisons with the diagonal pMGE5 area. Nevertheless, though we saw little histologic evidence of a pMGE1 origin of Sst cells, our results do not allow us to exclude categorically that some Sst cells may arise at this pallidal subarea.After initial experimental reports showed that the MGE gives rise to PV and SST cortical interneurons . Appropriate tests should be designed to check these possibilities. In any case, taken at face value, these results suggest that SST cortical interneurons are produced both at the dorsal and ventral Pal, that is at the pMGE1 and pMGE5 domains, thus providing partial support for our results. This suggests that our negative data about Sst cells originating within the Pal, or their absence inside the early Pal mantle layer, might be caused by late transcription of the Sst gene in the pMGE1-derived elements.Wonders et al.  dissociaNkx6.2 is expressed at the border between striatal and pallidal domains. It is specifically observed in a small subset of neural progenitors localized across the striatal progenitor domain pLGE4 and the pallidal progenitor domain pMGE1 . This generates the possibility that most SST cells are derived from other pallidal domains, including the Dg. When Nkx6.2-Cre/Nkx2.1-Cre/R26R-GFP double transgenic mice  were studied, the majority of SST+ cells  were marked with GFP. Since Nkx2.1Cre activity is practically absent at the pMGE1 domain  in Lhx6−/− mutants :TaumGFP brains. This analysis revealed that 10 and 23 % of Nkx2.1Cre:TaumGFP and 20 and 24 % of Gli1CreER(T2):TaumGFP recombined cells coexpressed SST at the medial posterodorsal and medial posteroventral nuclei  of the amygdala, respectively. This proportion was reduced to 2 and 3 % of cells coexpressing SST and ShhCre:TaumGFP in the MePD and MePV, respectively . They observed that the globus pallidus cell population was substantially eliminated, whereas most cortical and striatal interneurons were generated, excepting the striatal cholinergic neurons. In their Discussion, the authors deduce that pMGE1-3  generate most parvalbumin+ and somatostatin+ neo-cortical and hippocampal interneurons, because these domains do not express Shh. According to our interpretation, the Sst cells originated from the Dg ventricular domain—the pMGE5 area—may have partly escaped the targetted knock-out, since Shh expression only appears there in a patchy fashion . A 40 % of reduction in cortical SST+ cells was observed in the ShhCre-Nkx2.1-floxed mutant ; the missing or reduced globus pallidus phenotype may be due to a strong knock-out effect at the POA1 area, whose ventricular zone expresses strongly Shh and produces Shh-positive neurons that migrate into the pallidal mantle. These migrated cells may release SHH, which might be required for the normal development of the globus pallidus .Flandin et al.  used Shhne Figs. , S1; thine Figs. . The repSst cells, consistent with our Dg area, highlighted in our in situ analysis as the main origin of this interneuronal type. Our study emphasizes the supra- and subcapsular histogenetic and topographic unity of the Dg domain along the septoamygdaloid axis, where various radially migrated derivatives are established , and from where laterally oriented tangential migrations proceed into the striatum and the whole pallium . Several of the cited studies proposed that GABAergic interneurons from the MGE (including SST cells) first invade the pallium around E12.5 , POA2  and POH  . In the chick and mouse, the pPOA2 area expresses Nkx2.1, but not Shh  and approximately 25 % contained SST. They deduced that both PV and SST cells are produced at the pPOA2 area. This observation is contradictory with our observations, because we never saw Sst cells arising within the preoptic area. We would thus predict that the SST elements observed by Gelman et al. (Dbx1 mapped at E11.5 in the Allen Developing Mouse Brain Atlas (http://www.developingmouse.brain-map.org); the available sagittal and coronal sections both illustrate that at this stage the expression domain of Dbx1 extends more importantly across Dg than across the preoptic area. Part of the Dbx1-derived cells may be thus diagonal in character, rather than preoptic. Gelman et al. (2011) further acknowledged that permanent tracing of Dbx1-derived cells in Dbx1Cre; ROSA26YFP embryos generated ‘a few clones of YFP-expressing cells… in the MGE’. The authors checked the relationship of Dbx1-derived cells with Lhx6-derived cells  confusion in the denomination and/or tracing of the relevant progenitor areas, 2) experimental study mainly of suboptimal stages  which allow already migrated, or migrating neurons, to be ascribed to a wrong source, and 3) insufficient use (or dearth) of efficient molecular delimitation criteria for the Pal, Dg and POA subpallial domains and their diverse septoamygdaloid sectors. The present descriptive analysis cannot correct all these problems, but provides a solid basis of data that must be made consistent with any experimental analysis, as long as the existence of invisible Fig.S1 - Rostrocaudal series of topologically transversal cryostat sections through the MGE  at E10.5, illustrating in correlative adjacent sections the topography of Sst cells relative to Shh and Lhx7-8 expression: (a-f) Sst; (g-l) Shh; (m-r) Lhx7-8. Early Sst cells are observed close to the Dg ventricular zone . They aggregate at the marginal pallidal stratum and start to invade the striatum . The Dg ventricular zone shows patchy Shh expression (g-l). Lhx7-8 cells are clearly produced both at the pallidum and diagonal domains, but apparently not at the preoptic area (n-r) (TIFF 17717 kb)Fig.S2 – Sagittal section through the telencephalic subpallium in an E14.5 embryo (a) and two details at higher magnification, illustrating the topographic relationship of the diagonal Sst-positive supracapsular periventricular BSTM derivatives with DLX5 (b) and OTP (c) immunoreaction (superposed images from adjacent sections). (a) White dash lines delimit the St, Pal and Dg domains. The marker DLX5 labels uniformly the striatum, pallidum and diagonal, with a sharp boundary relative to the underlying alar peduncular hypothalamus (PHy), traversed vertically by the fibers of the cerebral peduncle (ped). The supracapsular BSTM (BSTMsc) is strongly Sst-positive, and lies at the border of the DLX5-positive field (detail at higher magnification in b). (c) shows a digital superposition of the Sst image with an adjacent section immunoreacted for OTP; this labels a distinct periventricular OTP-positive population that corresponds to the supracapsular migratory stream of Otp cells that move from the paraventricular alar hypothalamic area towards the medial amygdala; the comparison indicates that the latter migration courses outside the subpallium, that is, penetrates directly the pallium at the telencephalic stalk, neighboring the Dg domain (TIFF 6885 kb)Fig.S3 - Sagittal (a) and horizontal (b) sections through the adult mouse telencephalon, reacted by ISH for Sst, and counterstained with tyrosine hydroxylase immunoreaction (TH), illustrating Sst cell distribution in the striatum, olfactory cortex, hippocampus and the amygdaloid area, where the CeL subnucleus of CA stands out by its dense somatostatinergic population. This is held to derive radially from the amygdaloid sector of the Dg subpallial domain, jointly with the amygdaloid nucleus of the BSTM complex . There are sparser populations in the medial and capsular parts of the CA . Note also a dispersed population of Sst cells in the striatum, while Sst cells in layer III of the olfactory cortex (PirCx) are abundant , similarly as in the medial amygdala (MA), basomedial nucleus (BM) and lateral amygdalar nuclesu (L). In contrast, the basolateral nucleus remains poorly populated by Sst cells . Finally, note hippocampal Sst cells in a, mainly at the subiculum (Sub), alveus of field CA1, stratum oriens, pyramidal layer and stratum lucidum of CA3, and the dentate hylus (DG) (TIFF 8203 kb)Fig.S4 – Plate illustrating RC2 immunoreactive radial glia in the stria terminalis region (st) of an E18.5 mouse embryo (80 μm-thick Vibratome sections). The section plane is oblique , and courses along the septoamygdaloid axis , in order to intersect longitudinally the stria terminalis tract . Three adjacent section planes are shown (a-c), which progress from medial (Dg domain) to lateral . Reference landmarks visible include the anterior commissure , the internal capsule , the diagonal band (db) and the nucleus of the lateral olfactory tract (NLOT). Pannels  show higher magnification views of the boxed areas in b and c. The central amygdalar region lies under the amygdalar end of the stria terminalis, accompanied by the amygdalar BST nucleus . Radial glia fibres can be seen to originate at the ventricular lining , traverse vertically or obliquely the Ce field and end subpially in the neighborhood of the NLOT (other arrows in b-e). Some glial fibers are stretched along the stria terminalis , probably a passive deformation caused by the growth of the internal capsule, and recuperate a vertical orientation as soon as they reach the Ce region. Radial glia fibres pertaining to the pallial amygdala adopt slightly different courses  (TIFF 16425 kb)Below is the link to the electronic supplementary material.


Solanum lycopersicum L.) is an important vegetable crop and often cultivated in regions exposed to salinity and high temperatures (HT) which change plant architecture, decrease canopy light interception and disturb physiological functions. However, the long-term effects of salinity and HT combination (S+HT) on plant growth are still unclear. A dynamic functional-structural plant model (FSPM) of tomato was parameterized and evaluated for different levels of S+HT combinations. The evaluated model was used to quantify the contributions of morphological changes  and physiological disturbances  on the reduction of shoot dry mass under S+HT. The model predicted architectural variables with high accuracy (>85%), which ensured the reliability of the model analyses. HT enhanced architectural effects but reduced non-architectural effects of salinity on dry mass production. The stronger architectural effects of salinity under HT could not be counterbalanced by the smaller non-architectural effects. Therefore, long-term influences of HT on shoot dry mass under salinity were negative at the whole plant level. Our model analysis highlights the importance of plant architecture at canopy level in studying the plant responses to the environments and shows the merits of dynamic FSPMs as heuristic tools.Tomato ( Combining the knowledge from these studies, it seems that HT aggravates the architectural effects of salinity but ameliorates the non-architectural effects. However, the magnitudes of these aggravation and amelioration and the long-term effects (more than weeks) of them on dry mass production at the whole plant level are still unknown. The aim of this paper is to test the hypothesis that HT increases architectural effects of salinity but reduces non-architectural effects of salinity by combining data from five experiments and analyses from a dynamic FSPM. Furthermore, the sensitivity of dry mass production to architectural parameters in the FSPM was analyzed to investigate the importance of architectural traits on stress tolerance.Salinity is often associated with high temperatures HT; . It is slpfg plant modeling program  grown under non-saline conditions. To implement the effects of salinity on plant architecture into the model, the same tomato cultivar was used in four experiments conducted in the growth chambers (Experiments 1–3) and greenhouses (Experiment 4) of Leibniz Universität Hannover, Germany. Experimental details can be found in the Supplementary Table S2.The dynamic FSPM of The growth chamber experiments were designed to investigate the environmental effects  on leaf and internode responses to salinity . In these experiments, leaf length (cm) and internode length (cm) were measured by a ruler daily. The growth chamber experiments were set up with four levels of salinity  in combination with a variation of temperature, VPD and PAR. In the growth chambers, the experiments were arranged in split plot designs with environmental conditions as main plot factors and salt stress as the sub plot factor.Experiment 4 was conducted in 2009 and was used to examine the effects of salinity on leaf shape, leaf angle, and curvature , which were derived from weekly plant digitizing using a Fastrak 3D digitizer . In Experiment 4, salinity treatments (for 4 weeks) were identical with those in Experiments 2 and 3 and air temperature, VPD and PAR in the greenhouses were recorded hourly. The greenhouse experiment was set up as a randomized complete block design with four replications and four plants per plot. Plant protection was applied when necessary.To evaluate the model for tomato architecture under salinity stress, Experiment 5 was established in spring of 2010 in two greenhouses with 22/18 and 32/28°C day/night temperature, representing low temperature (LT) and HT conditions, respectively. Salinity treatments in Experiment 5 were identical with those in Experiments 2–4 and were applied on 21 day after the first true leaf appearance (DAFLA) for 7 weeks. In this experiment, leaf and internode length, leaf number, and plant height were recorded twice a week and the whole plant architectures were recorded by a Fastrak 3D digitizer weekly as in Experiment 4. Four plants per treatment were harvested on 28, 35, 43, 50, 56, 63, 70, and 77 DAFLA. Leaf area of the harvested plants were measured using a leaf area meter  and then dried at 70°C for at least 96 h to determine dry mass. Further details of the experimental set-ups, cultivation schedule and weather data of the greenhouse experiments can be found in Supplementary Table S2 and in Simulations started from the DAFLA and were run for 80 days under two different temperature regimes with the measured climate data from Experiment 5 and under four salinity levels . Simulations were run five times with a randomized variation in phyllotaxis angle (144 ± 10°). At the organ level, measured and simulated leaf and internode growth over time was compared for rank 8. At the canopy level, measured and simulated leaf number, plant height , whole plant leaf area and shoot dry mass  were compared. Simulated and measured data were compared using root mean square deviation (RMSD), bias and accuracy .t, ΔWl(t) (g d-1), was the product of leaf area (Al(t), m2), light absorption of the leaf  and light use efficiency, 𝜀 (g CO2 J-1):Dry mass production by a leaf at time Iabs(t)) is an empirical light-dependent function for tomato derived from kT,x is the effect of temperature and salinity on light use efficiency . Daily increase in plant dry weight  was the integration of ΔWl produced by all leaves and the plant dry weight (Wp(t), g) was then the integration of ΔWp with time. The shoot weight (Wsh(t), g), was considered a constant proportion of Wp(t) . To predict canopy dry mass production under various environmental conditions, using canopy light use efficiency has been demonstrated to be a robust approach . Here, we present a new method to estimate the changes in kT,x during the growing period using the dynamic FSPM and measured allometric relationships between shoot dry mass and total leaf area.where 𝜀(approach . Furtherapproach . In Eq. An) and shoot dry mass under non-stress conditions, Wsh,n, were described by:Allometric relationships between plant traits have been shown from cell to population levels . For exapn and qn are empirical coefficients for non-stress conditions. Since salinity slightly changes the allometric relationship between total leaf area and shoot dry mass :where dry mass , coefficp) and intercept (q) parameters, are influenced by temperature  leaf growth dynamics, (2) leaf distribution in the space, and (3) canopy light use efficiency. Our model predicts leaf growth dynamics and leaf distribution with high accuracies ) in Eq. 1], which can be influenced by leaf age, temperature :A crop model where perature  and bothperature , 2008. TWsh,m and Wsh,r are the shoot dry mass at time t based on the measured allometric relationships  and simulations with reference canopy light use efficiency, respectively. The steps for time t were 28, 35, 43, 50, 56, 63, 70, and 77 DAFLA. By running the model for unstressed conditions with kT,0⋅𝜀(Iabs(t)) instead of 𝜀(Iabs(t)), the simulated allometric relationships between total leaf area and shoot dry mass should fit the measured relationships (Eq. 2a). The same, by running the model for stress conditions with kT,x⋅𝜀(Iabs(t)), the simulated allometric relationships should match Eq. 2b. Here we want to emphasize that the biological meaning of kT,x is the relative photosynthetic capacity of a whole plant, an outcome of combined effects of temperature, salinity, leaf and canopy age. For this reason, temperature and salinity effects on relative canopy light use efficiency were further dissected:where kLT,0 is the relative canopy light use efficiency under LT and non-salinity condition, kHT is the effects of HT (set to 1 for LT conditions) and kx is the effects of x mM NaCl in the nutrient solution (set to 1 for 0 mM NaCl).where Ra,x and Rn,x, respectively, %) at x mM NaCl was calculated by:The architectural and non-architectural effects of salinity on dry mass production ; Wsh,a is the shoot dry mass simulated with the architecture being affected by x mM NaCl but with the control light use efficiency; and Wsh,x is the shoot dry mass simulated with architecture and light use efficiency affected by x mM NaCl. The term Wsh,0 – Wsh,a in Eq. 4a represents the difference in shoot dry mass resulted from salinity effects on total leaf area, leaf angle, and canopy light interception, the architectural effects. The term Wsh,a – Wsh,x in Eq. 4b is the reduction of shoot dry mass due to the salinity effects on light use efficiency, the non-architectural effect. where x was used for quantifying the effects of architectural traits on light interception and dry mass production under 40 and 80 mM NaCl, separately for both temperature regimes. Leaf number, leaf area, internode length and leaf angle were chosen for the analyses because they are most frequently reported to be influenced by salinity. The testing range for each trait was determined according to the values reported in the literature (Supplementary Table S1): the reduction of leaf number was by 3, 6, and 9% per 10 mM NaCl in the solution. Reduction of internode length was by 2, 4, and 6% per 10 mM NaCl in the solution. To evaluate this effect of salinity on leaf area, sensitivity of leaf elongation rate to salinity  was simulated with 50–150% of the reference value. Leaf angle was simulated with 70–130% of the reference values (100%). Only one morphological trait was changed for each analysis.The FSPM was used to test the sensitivity of dry mass production to architectural parameters under salinity. The model with light use efficiency equal to 𝜀R2 = 0.71, data not shown) and both of them influenced the leaf elongation (Eq. S2). In Experiment 5, reduction in shoot dry mass due to salinity stress under HT was stronger than under LT on day 77 DAFLA. In comparison with the tomato plants grown under control conditions, measured shoot dry mass of plants grown under 40, 60, and 80 mM NaCl was reduced by 6.1, 22.5, and 28.6%, respectively, under LT, and 19.9, 30.3, and 39.4%, under HT conditions . On day 77 DAFLA, total leaf number and plant height were not different between salinity treatments (data not shown).It is important to stress that the temperature effects presented in this study were the combined effects of temperature and VPD since they were highly correlated in the experiments . At HT, final leaf length was underestimated by 2.7–4.8 cm . Predicted leaf lengths had accuracies higher than 85% (Table 1). Salinity had no effect on internode length for both LT and HT  and the model overestimated the leaf length under LT and internode growth in the early phase. This resulted in lower accuracies in predicting internode length (Table 1). However, standard deviations of the measured final internode lengths were high and the difference between measured and simulated final internode lengths were less than 1 cm .The model described the reduction of leaf length due to salinity under LT very well  and HT . For all salinity levels and both temperature conditions, the simulated total leaf area was well in accordance with the measurements . The measured shoot dry mass under 80 mM NaCl was 23% less than under 40 mM NaCl at LT but, interestingly, the simulated 16% reduction of total leaf area under LT  reduced the simulated dry mass production by only 1.1% . In contrast, the simulated shoot dry mass under 80 mM NaCl was 13% less than under 40 mM NaCl at HT . The accuracies of the simulated shoot dry mass with reference canopy light use efficiency (Eq. 1) decreased with the salinity level for both LT and HT conditions (Table 1). The random factor in the model only resulted in a very slight difference (<1%) between simulations. Therefore, the simulated data shown in Figures 2 and 3 were the results of one simulation.Both measured and simulated results show that salinity reduced total leaf area under LT . Running the model with canopy light use efficiency equal to kT,x⋅𝜀(Iabs) instead of the reference light use efficiency, the simulated allometric relationships matched the measured relationships .Significant allometric relationships between total shoot dry mass and total leaf area were found  was higher than 1 for days 29–56 and decreased with time (Table 2). Furthermore, HT reduced canopy light use efficiency, kHT. Light use efficiency decreased with the increasing salinity level under both LT and HT condition (k40 < k60 < k80) and with time after exposure to salinity (Table 2). The degree of this decrease with time under 60 and 80 mM was stronger than under 40 mM NaCl.Under LT conditions, the relative canopy light use efficiency  decreased with time after exposure to salinity (Table 3). Architectural effects depended on temperature regimes and increased with salinity level. In general, they were stronger at HT than at LT. For example, architectural effects at 80 mM NaCl over the whole growing period reduced dry mass production by 9.7 and 21.9% under LT and HT, respectively. Furthermore, architectural effects did not change strongly with salinity level under LT conditions while under HT they were twice as high as at 80 than at 40 mM NaCl (Table 3).The reduction (%) in total leaf area under salinity was similar between LT and HT conditions (data not shown). Both under LT and HT conditions, the architectural effects on reducing dry mass production  increased with time and were higher at LT than at HT for the whole growing period. Both architectural and non-architectural effects increased with salinity level. They were close to zero under 40 mM NaCl and increased to 29 and 19% under 80 mM NaCl at LT and HT, respectively. Furthermore, the sum of architectural and non-architectural effects was similar to the measured reduction of shoot dry mass under salinity .Non-architectural effects increased with salinity level. In contrast to architectural effects, non-architectural effects (Table 4). The sensitivity of dry mass production to architectural traits was temperature dependent. For example, while the change in leaf elongation rate  influenced the total leaf area in the same magnitude at both LT and HT conditions, shoot dry mass at LT was not affected but reduced by up to 20% at HT (Table 4). Shoot dry mass was less sensitive to internode length and leaf angle at HT than at LT. Changes in leaf angle had less influence on dry mass production under 80 mM NaCl than under 40 mM NaCl. The reduction of dry mass production was linearly related to the light interception by the canopy . The change in light interception explained 85 and 76% of the reduction in dry mass production at LT and HT, respectively. Furthermore, both at LT and HT conditions, the sensitivity of shoot dry mass to internode length was similar at 0, 40, and 80 mM NaCl (Supplementary Table S3).Shoot dry mass was most sensitive to leaf number and the reduction of leaf number decreased total leaf area almost linearly (Table 3).The results from our model analyses support the hypothesis that (1) architectural effects of salinity are more prominent under HT than under LT, especially under high salinity and (2) non-architectural effects are more prominent under LT than under HT (Table 3). This indicates that architectural effects of salinity are stronger in a younger open canopy and decrease with canopy closure, similar to the impacts of architectural traits on dry mass production under non-stressed conditions  and were more closed also explains why architectural effects of salinity are smaller under LT than under HL. Interestingly, under LT condition, the increase of salinity level from 40 to 80 mM NaCl reduced the total leaf area by additional 20%  but this reduction in leaf area only resulted in an extra 0.9% of architectural effects on dry mass production (Table 3). This could be explained by the fact that light interception of the canopies under 40 and 80 mM NaCl were about equal , indicating that the architectural effects of salinity at LT were mainly an effect of leaf angle, but not of leaf area. This is also the reason why shoot dry weight was less sensitive to the leaf elongation  under LT (Table 4).The primary architectural effects of salinity are the reduction of leaf area and leaf angle . The rednditions . The fac+ uptake rate of tomato plant is reduced under HT . The simulated results from a model, where the leaf growth dynamics, distribution of leaves in the space, light interception and photosynthesis are described precisely, should also be able to describe these allometric relationships. Since we have carefully evaluated our architectural model and shown that both at organ and canopy levels, our architectural model may predict the dynamic changes of plant architecture with very high accuracies  based on the measured allometric relationships between shoot dry mass and total leaf area. To assure that these estimations are plausible, we carefully examined the prerequisites and the results of this new method.The measured data showed strong allometric relationships between shoot dry weight and total leaf area .Very importantly, we want to emphasize the prerequisite of this method. Model analyses have shown that dry mass production can be strongly influenced by architectural traits while the simulated leaf area maintains the same . This innd roots . Form oukT,x, and effects of HT and salinity on it, kHT and kx, respectively,  showed several trends: (1) kHT was smaller than one; (2) kLT,0 and kHT,0 decreased with time; (3) kx decreased with increasing salinity level; (4) kx decreased with time after exposure to salinity; (5) the magnitude of the decrease in kx with time under 60 and 80 mM NaCl was stronger than it under 40 mM NaCl; and (6) kx under LT, especially under 80 mM NaCl, was smaller than it under HT. Because that all this trends can be well-explained by the findings reported in the literature, we consider that our estimations of the relative canopy light use efficiency and the following quantification of architectural and non-architectural effects were plausible. First, under control conditions, tomato has its best photosynthetic performance at around 25°C ) in Eq. 1] was parameterized from a mature tomato canopy . Furthermore, the magnitude of the decrease in kx with time under 80 mM NaCl was stronger than it under 40 mM NaCl. This indicates that the ionic effect appeared faster and was more prominent under higher salinity. Finally, that kx under LT was smaller than it under HT can be explained by the recent finding that tomato grown under LT accumulates more Na+ in leaves than grown under HT . This indicates that maintaining young leaf production under salinity stress is a key architectural trait for salinity tolerance. Furthermore, maintaining young leaf production may also counterbalance the leaf senescence due to the ionic effect (Table 4). This would partly explain the negative relationship between salt tolerance and salt-induced increases in leaf angle (Table 4). The light interception by the canopy may explain 85% of the effects of leaf angle on shoot dry mass while no relationship was found between the effects of internode on light interception and on shoot dry weight . Reduction in internode length decreases the distance between leaves and enhances the self-shading (The decrease in leaf appearance rate and leaf number under salinity had the strongest effects on reducing total leaf area and dry mass production (c effect . The chaaf angle . Changes-shading , which r-shading .High temperatures aggravate the negative effects of salinity on dry mass production via plant architecture and light interception but ameliorate the salinity effects on canopy light use efficiency. These results highlight the different temperature effects on physiological and morphological responses to salinity and the importance of plant architecture in studying plant responses to environmental changes at canopy level. Our analyses suggest that leaf angles influence light interception more than light distribution, and that changes in internode length have stronger effects on light distribution than on light interception. Furthermore, our model analyses enable us to dissect the architectural effect from non-architectural effect of salinity, which is impossible to be done experimentally because in reality both effects occur together under osmotic stress.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A number of root and root hair traits have been proposed as important for nutrient acquisition. However, there is still a need for knowledge on which traits are most important in determining macro- and micronutrient uptake at low soil fertility. This study investigated the variations in root growth vigor and root hair length (RHL) and density (RHD) among spring wheat genotypes and their relationship to nutrient concentrations and uptake during early growth. Six spring wheat genotypes were grown in a soil with low nutrient availability. The root and root hair traits as well as the concentration and content of macro- and micronutrients were identified. A significant genetic variability in root and root hair traits as well as nutrient uptake was found. Fast and early root proliferation and long and dense root hairs enhanced uptake of macro- and micronutrients under low soil nutrient availability. Vigorous root growth, however, was a better indicator of early nutrient acquisition than RHL and RHD. Vigorous root growth and long and dense root hairs ensured efficient acquisition of macro- and micronutrients during early growth and a high root length to shoot dry matter ratio favored high macronutrient concentrations in the shoots, which is assumed to be important for later plant development. Nutrient availability is the primary limitation to nutrient uptake and crop productivity in low-input and organic agriculture  genotypes representing both recent and older genotypes to low P concentration under hydroponic growth conditions 2·6H2O, 0.9 mM Ca (NO3)2·4H2O, 0.6 mM KNO3, 1 μM MnCl2·4H2O, 0.8 μM Na2MoO4, 0.7 μM ZnCl2, 0.8 μM CuSO4·5H2O, 2 μM H2BO3, 1 μM NiSO4·6H2O, and 50 μM Fe-EDTA. The culture solutions were continually aerated and their pH was maintained between 5.8 and 6.0 with daily additions of KOH and HCl. Based on their root length and RHL after 24 days growth at P limiting (2 μM P) condition , available K 57 mg kg−1 (ammonium acetate extractable), available Ca 798 mg kg−1 (ammonium chloride extractable), available Mg 30 mg kg−1 (ammonium acetate extractable), available Mn 5 mg kg−1 (magnesium nitrate extractable), available Cu 17 mg kg−1 (EDTA extractable), available Zn 15 mg kg−1 (EDTA extractable), and available B 45 mg kg−1 (EDTA extractable).Soil for the experiment was collected from the upper horizon (0–30 cm) in a long-term field trial, from a treatment which has not been fertilized but grown with cereals including undersown clover grass mixture for green manure since 2003 at the experimental farm of University of Copenhagen  fertilizer at the rate of 100 kg N ha−1 was applied and mixed homogeneously into the entire volume of soil and sand mixture in all the tubes. Two plants were grown in each tube, and each genotype was replicated four times. The tubes were watered to soil water holding capacity initially and then watered to 80% of soil water holding capacity twice a week. The seeds germinated on June 12th 2012, and the plants were harvested on July 12th, 2012 [30 days after emergence (DAE)] at stem elongation growth stage. The soil columns were excavated into a water bath and then all the roots were washed out using a gentle stream of water with great care to minimize any potential damage to root hairs.Nitrogen  and analyzed by the WinRHIZO image analysis system  for root length and surface area. Randomly selected root segments in the root hair zone from each replicate were placed in a film of water in petri dishes. Root hair images were captured for all the genotypes on the main root axis and first order and second order roots, using a video camera fitted to a microscope  at 2.5 × magnification interfaced with a computer image grabber board. Root hairs were analyzed using ImageJ software. All root hairs were measured manually with the same procedure. Root hair length (RHL) was measured at more than 100 randomly selected root hairs for each replicate. Root hair density (RHD) was determined as the number of root hairs per mm root length. The total length of root hairs mm−1 root (TLRH) was calculated by multiplying RHL and RHD.Transparent plastic sheets with printed grid lines were placed on the surface of the Perspex tubes to measure n et al.  and expr3 and HCl in a 1:3 v:v mixture, then analyzed for P, K, Ca, Mg, S, Fe, Mn, Cu, Zn, and B using inductively coupled plasma-optical emission spectroscopy . Total N was analyzed using the Dumas dry combustion method in a system consisting of an ANCA-SL Elemental Analyser coupled to a 20–20 Mass Spectrometer . Nutrient content was calculated as multiplication of concentration by dry matter with subtraction of seed reserve.The DM of shoot, root and original seed material was determined after oven drying to constant weight at 70°C. After grinding, the samples were digested in a microwave oven with HNOThe data were analyzed by one-way analysis of variance (ANOVA) using SAS GLM  at a significance level of 5%. Duncan's multiple range test was applied to assess the differences between treatments at a significance level of 5%. Linear regression analyses were used to determine the relationships between the measured parameters.The root development over time differed markedly among genotypes Figure , with ApSignificant differences in RHL, RHD and TLRH among genotypes were also observed. The roots of April Bearded and Hindy62 were covered with the longest and densest root hairs, whereas Farah had the lowest RHL, RHD and TLRH Figure .There were substantial and significant differences in shoot DM among genotypes Table . Shoot DThe concentrations of macro- and micronutrients in the shoots showed remarkable differences among genotypes, the only exception being sulfur (S) with no significant difference. We analyzed the correlations between root length or root hair traits and nutrient concentrations, but no such correlations were found. However, concentrations of N, P, K, Ca, and Mg were higher in Farah, April Bearded, Hindy62 and Dacke compared to A35–213 and Hankkijan Tapio, except for lower Ca in April Bearded and lower Mg in Farah  root length and macro- and micronutrient content, and root surface area and macro- and micronutrient content, with the exception of B Table ; (2) RHLr2 = 0.85, P < 0.01) across macronutrients, but not significant across micronutrients .We examined the average nutrient concentrations of genotypes against their respective root length/shoot DM ratio. This may be seen as an indicator of the balance between root supply and shoot demand. For each nutrient the concentration of each genotype relative to the average concentration across the six genotypes was calculated. This value was then averaged across the six macronutrients of N, P, K, Ca, Mg, and S or five micronutrients of Fe, Mn, Cu, Zn, and B Figure . The resRoot traits influencing the acquisition of mineral elements include root length, root hair characteristics, and root distribution in soil layers, all of which increase the volume of soil explored by the root system and the surface area for uptake of mineral nutrients (White et al., Genotypes used in the current study showed significant differences in root distribution and depth during the experimental period, which may contribute to differences in nutrient and water uptake Gregory, . While nHigher macronutrient concentrations were related to a higher ratio of root length to shoot DM Figure , indicatHowever, while it is clear that a vigorous root growth can enable a more rapid depletion of nutrients in a low fertility soil, a number of other traits could be important for determining the genetic variability in the efficiency of nutrient uptake of the root system. Differences between cultivars could also be due to differences in the excretion of protons, organic acids and other exudates Lynch, .Nutrient uptake is dependent on one hand on root nutrient uptake capacity by the root system but also on above-ground growth creating shoot nutrient demand. If nutrient uptake is stimulated by increased shoot demand without a concomitant increase in supply from roots, this will lead to reduced nutrient concentration in the plant due to dilution; this effect can be seen in Hankkijan Tapio and Dacke—where Hankkijan Tapio has higher shoot dry matter and total uptake of most nutrients but simultaneously lower concentrations Figure , yet DacOur results show clearly that there was a significant genetic variability in root and root hair traits under low soil nutrient availability. A high root length to shoot dry matter ratio favored high concentration of macronutrients in the shoots. Vigorous root growth and long and dense root hairs are important traits in ensuring efficient acquisition of both macro- and micronutrients in the early establishment of spring wheat in nutrient-limited soil and low nutrient input cropping systems.JM, KT, and LS formulated the research questions. YW, JM, KT, and LS designed the experiment. YW performed the experiments, analyzed the data and drafted the manuscript. JM, KT, and LS participated in the data analysis, assisted with the revisions to the manuscript and coordination of the study.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer JK and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Twenty five healthy controls were group matched for age and gender whose BMI was 16-25 kg/m2. Serum S100B and leptin levels and positive and negative symptom scale (PANSS) were assessed at admission and after six weeks. During the study, S100B showed a strong and negative correlation with leptin . Also, there were negative correlation between serum S100B level and PANSS negative subscale after 6 weeks of treatment .Recently, extensive efforts have been made to understand the rate of energy expenditure and the weight gain associated with atypical antipsychotic treatment, including identification of markers of obesity risk. In recent years, leptin, an adipocyte hormone, has gained significant interest in psychiatric disorders. S100B has been considered as a surrogate marker for astrocyte-specific damage in neurologic disorders. Also, S100B has been detected in adipose with concentration as high as nervous tissue as a second release source. In this study we evaluated the relationship between S100B and leptin in schizophrenic patients under treatment with clozapine and risperidone.This study included 19 patients meeting the DSM-IV-TR criteria for schizophrenia, having body mass index (BMI) of 16- 25 kg/mPositive correlation between leptin level and PANSS suggested a potential role for leptin which can mediate the link between antipsychotic induced weight gain and therapeutic response in schizophrenia. Schizophrenia is a serious disorder with a worldwide prevalence of about one percent and is associated with a 20% higher mortality rate compared to the general population . Antipsy+2 homeostasis, transcription factors and glucose metabolism  in Sari in north of Iran. It was approved by the ethic committee of Mazandaran University of Medical Sciences (MAZUMS) and written informed consent was obtained from all participants’ guardians.Patients2 and suffering schizophrenia for more than 3 years. During the period from September 2010 and November 2012, eighty five patients with schizophrenia were registered into the trial but forty met the inclusion criteria. Subjects were included in the study if meeting DSM-IV-TR criteria  for schi2 as control group from general population for establishment of normal serum levels of S100B and leptin.The schizophrenia patients were compared with 25 age- and gender-matched healthy volunteers Who had BMIs of 16 to 25 kg/mPatients with other psychiatric disorders, a prior history of neurologic disorders, acute or chronic illnesses known to affect the immune, endocrine or metabolic system like pulmonary, infectious, and coronary heart diseases, neoplasm, manifested diabetes, hyperlipidemia, history of substance abuse or dependence, dementia, severe trauma, suicide attempts, a previous history of cholesterol lowering treatment and with alimentary restriction or evidence of clinical malnutrition were excluded from the study. In control group, the aforementioned disorders and psychiatric disorders were excluded after taking a detailed history. The subjects did not take any concomitant medication. Thirteen patients were on risperidone and six on clozapine, for at least 6 months  at the time of blood sampling. During the study period only co medication with anticholinergic drugs and benzodiazepines were allowed. Fourteen subjects suffered from undifferentiated schizophrenia, four from paranoid schizophrenia and one from residual schizophrenia. All patients had same food regime during the study. All schizophrenic patients were managed based on the guideline of the American Psychiatric Association . One psySample collectionBiomarker measurementBlood was drawn after an overnight fast by venous puncture at 7 A.M on admission and after six weeks. Each sample was centrifuged (3500 × g) for 15 min then the serum was separated and stored at -80˚C for further analyses. A complete differential blood cell count, including total cholesterol, triglyceride, LDL-cholesterol, HDL- cholesterol levels, fast blood sugar, systolic and diastolic blood pressure, and BMI were measured on admission and also after six weeks for all patients.Serum S100B was analyzed using commercially available ELISA kit  according to the manufacturer instruction.Leptin levels were determined by an enzyme linked immunosorbent assay kit  according to the manufacturer instruction.All samples were assayed in duplicate. Statistical analysisAll data were assessed for normality by one sample Kolmogorov-Smirnov test. Qualitative variables were recorded in frequency and percentage and quantitative variables in Mean ± SD (Standard Deviation). To compare continuous variables in two independent groups, we used t-test or Mann-Whitney U Test. Chi-Square test was applied to compare categorical variables. Also, paired t-test was used to compare two related continuous variables. The correlation between quantitative variables was made by spearman test. All statistical analyses were conducted using SPSS version 18  and P value of less than 0.05 was considered significant.During the period from September 2010 and November 2012, eighty five patients with schizophrenia were registered into the trial but forty met the inclusion criteria. Forty patients who met the inclusion criteria entered the study, of which three did not complete their study, six were misdiagnosed .Among all the patients who received antipsychotic drugs, eight patients, psychiatrists changed their antipsychotic drug regimen and four received antipsychotic combination therapy during the study .Finally nineteen patients completed the study . Baseline demographic and other baseline measurements between the study and control group are shown in On admission, the lipid profiles in matched healthy controls were significantly higher compared to those of the schizophrenic patients .The mean serum concentration of S100B and leptin were 26. 4 ± 16.8 picog/mL and 11.7 ± 17.2 microg/mL, respectively for the control group. Healthy control group had higher initial serum levels of S100B and leptin but not significant at admission  .The lipid profiles and FBS levels increased during the study period, but only elevation of triglyceride level was significant  . Our intThe correlation between changes of serum S100B, leptin, BMI, lipid profiles, FBS and PANSS  are illustrated in Table 3. During the study, S100B showed a strong and negative correlation with leptin . This significant prediction of S100B by leptin could be highly attributed to its close dependence on adipose tissue mass like S100B. Also, there were negative correlation between serum S100B level and PANSS negative subscale after 6 weeks of treatment cells in arcuate nucleus and leads to increase in the release of melanocortin peptides. This peptide inhibits food intake and regulates metabolism by energy storage and insulin secretion . Leptin et al  and glycogenolysis. Insulin has been shown to down regulate S100B expression in astrocyte cultures and rat brain with s100B binding to fructose-1, 6 bisphosphonate aldolase and phosphoglucomutase that may improve intracellular energy balance . In schiet al study who found a positive relationship between S100B and leptin levels. One possible reason could be that in their subjects S100B concentrations were particularly elevated at baseline and samples had BMI > 30. We found positive correlation between change in leptin level and PANSS which is in accordance to the studies of Venkatasubramanian G et al who suggested a potential role for leptin which can mediate the link between antipsychotic–induced weight gain and pleasure therapeutic response in schizophrenia  and explore metabolic parameters (leptin and insulin levels) in both human serum and cerebrospinal fluid .Declaration of conflicting interests: None declared. Funding Acknowledgements:This research was financially supported by Mazandaran University of Medical Sciences grant.


CHS expression and the petal coloration level in different cultivars is still unclear. In this study, three typical crabapple cultivars were chosen based on different petal colors and coloration patterns. The two extreme color cultivars, ‘Royalty’ and ‘Flame’, have dark red and white petals respectively, while the intermediate cultivar ‘Radiant’ has pink petals. We detected the flavoniods accumulation and the expression levels of McCHS during petals expansion process in different cultivars. The results showed McCHS have their special expression patterns in each tested cultivars, and is responsible for the red coloration and color variation in crabapple petals, especially for color fade process in ‘Radiant’. Furthermore, tobacco plants constitutively expressing McCHS displayed a higher anthocyanins accumulation and a deeper red petal color compared with control untransformed lines. Moreover, the expression levels of several anthocyanin biosynthetic genes were higher in the transgenic McCHS overexpressing tobacco lines than in the control plants. A close relationship was observed between the expression of McCHS and the transcription factors McMYB4 and McMYB5 during petals development in different crabapple cultivars, suggesting that the expression of McCHS was regulated by these transcription factors. We conclude that the endogenous McCHS gene is a critical factor in the regulation of anthocyanin biosynthesis during petal coloration in Malus crabapple.Chalcone synthase is a key and often rate-limiting enzyme in the biosynthesis of anthocyanin pigments that accumulate in plant organs such as flowers and fruits, but the relationship between  Arabidopsis thalianaPetunia hybridaZea maysMalus domesticaCHS and its involvement in many aspects of plant physiology and biochemistry.The plant phenylpropanoid biosynthetic pathway leads to the formation of numerous compounds that are involved in diverse physiological and biochemical processes CHS gene was cloned from parsley (Petroselinum crispum) in 1983 CHS genes have been isolated, mostly from monocots and dicots, including the legume soybean (Glycine max), alfalfa (Medicago sativa), pea (Pisum sativum), A. thaliana, barley (Hordeum vulgare), corn (Zea mays), grape (Vitis vinifera) and others CHS genes has shown them to be ubiquitous in plants, including early land plants and algae of the Charophyceae The first CHS has been well studied in the context of the synthesis and accumulation of anthocyanin pigments and several reports have described the effects of altering CHS expression in transgenic plants. For example, expression of an antisense CHS gene in petunia resulted in flowers that were pale colored, or even white, due to an inhibition of anthocyanin production, and plant fertility was also affected CHS-silenced strawberry (Fragaria × ananassa) lines were reported to have a lighter pink/orange coloration and a significant decrease in the levels of all flavonols, proanthocyanidins and anthocyanins Malus domestica) CHS-RNAi knockout lines were shown to have no detectable anthocyanins accumulation and substantially reduced levels of dihydrochalcones and flavonoids Malus crabapple has one of the most economically important sets of ornamental apple germplasm resources. This represents a valuable source of research material, since crabapple exhibits excellent stress resistance and is useful for investigating the mechanism of plant pigmentation, due to the diversity of color in its leaves, flowers and fruits as a consequence of anthocyanins accumulation McCHS involved in the biosynthesis of anthocyanins and coloration level in the petals of these cultivars, we compared the expression of McCHS and other flavonoid biosynthesis pathway structural genes during petal expansion in these three typical cultivars by quantitative real-time PCR (qRT-PCR). Meanwhile, HPLC analysis provided an insight into the accumulation of anthocyanins and other flavonoids compounds in these different crabapple cultivars. The results suggest that the expression of McCHS in petals is well controlled, in a tissue- and developmentally specific fashion. We also overexpressed the McCHS gene in tobacco to evaluate its activity and the consequences of its expression. To sum up, McCHS expression is associated with petal coloration and the expression level of McCHS determined the coloration level of petals in different crabapple cultivars. Meanwhile, the expression level of this gene may be regulated by multiple MYB transcription factors.Malus crabapple cultivars, ‘Royalty’, ‘Radiant’ and ‘Flame’, are shown in The petal phenotypes of three Malus crabapple petal flavonoid composition and its variation among cultivars, the levels of two anthocyanins (cyanidin and pelargonidin) and five other flavonoids were measured in the petals of the three cultivars , a gene sequence for the full length CHS (McCHS) was amplified from total RNA of Malus crabapple ‘Royalty’ leaves by RT-PCR and RACE. Specifically, the McCHS cDNA (accession no. FJ599763) is 1,529 bp in length and is predicted to encode a protein of 389 amino acids with a high level of homology to with CHS genes from a range of species , Malus domestica , Malus domestica , Pyrus bretschneideri , Malus domestica  and so on. It appears that chalcone synthase protein sequences have undergone little sequence diversification in the species examined and that they are highly similar in length in different plant species. Other than the putative CHS gene sequences, there are also related sequences that are more divergent from characterized CHS genes and although these genes show high sequence similarity to a Fragaria ananassa CHS , e.g. Rubus idaeus , their functions have yet to be determined. Collectively they are described as polyketide synthase (PKS) genes, a more general description for the CHS and CHS-Like gene family  and apigenin. In ‘Flame’ petals, the expression of McCHS also showed a positive correlation with McMYB5 expression . Meanwhile, the expression trend of TFs and McCHS were consistent with the accumulation of kaempferol, quercetin, rutin, apigenin and catechin, which were the main flavonoids compounds in white petal cultivar ‘Flame’. These data are consistent with McMYB4, McMYB5 that may be involved in the regulation of McCHS expression during petal development in these cultivars, and these results suggested that due to different accmulation of flavonoids compounds, the McCHS gene have different biosynthesis functions and regulated by different transcription factors.McCHS in plants, transgenic tobacco plants expressing McCHS under the control of a constitutive CaMV35S promoter were generated, and two independent T2 lines were characterized. Compared to wild type, tobacco plants transformed with 35S::McCHS developed a more intense pigmentation in the petals, especially in McCHS-ox-1 line  decreased to one-third of that of control plant petals and the ‘a*’ values were 4.0 and 5.0-fold higher than in control plant petals, indicative of a stronger red color , including CHS gene McCHS has several MYB-binding cis-elements and maybe regulated by MYB TFs McCHS can be regulated by these transcription factors in different crabapple cultivars due to the various flavonoids compounds accumulation  resulted in an increase in total phenolic compounds in Linum usitatissimumCHS gene expression in several plants has been shown to result in reduced anthocyanin levels in flowers. CHS gene silencing in tobacco P. hybridaMalus crabapple.To confirm that the accumulation of flavonoids was influenced by expression of the CHS is regulated by several factors, such as environmental conditions, the variation in the conditions in plants (such as pH), transcription factors and so on OgCHS promoter altered the expression of this gene in different Oncidium orchid cultivars McCHS expression in crabapple.The expression of McCHS is consistent with the variation of petal color and anthocyanins accumulation in different crabapple cultivars, and the expression intensity of McCHS determined the petal pigmentation level in crabapple specially. On the other hand, the expression of McCHS is responsible for the color fade process during petal expansion in color variation cultivar. The regulation mechanism of McCHS expression is complicated, and several transcription factors might be involved in regulating McCHS expression in crabapple petals. Key future questions that need to be addressed include but not limited to the transcription regulation and epigenetic modification of McCHS expression. Undoubtedly the work described in this report will trigger a series of exciting future research projects to elucidate the mechanisms governing plant coloration.In this study, we have shown evidences that the expression level of Malus crabapple cv. ‘Royalty’, ‘Radiant’ and ‘Flame’ were collected at different development stages . These trees were grown in the Crabapple germplasm of Beijing University of Agriculture  received standard horticultural practices, and disease and insect control. Control and T2 transgenic tobacco (Nicotiana benthamiana) plants were grown in a greenhouse. Flowers were collected for studies at the full-bloom stage (IV). The leaves of Malus crabapple cv. ‘Royalty’, ‘Radiant’ and ‘Flame’ were collected at the same time with petals at the full bloom stage. Fruits were collected at their fully mature stage in October 2012. At sampling time all the plant materials were immediately frozen in liquid nitrogen and stored at -80°C until RNA or phenolic compounds were extracted.The petals of L*a*b* coordinate, namely lightness and hue, were measured immediately after flowers were picked with a hand spectrophotometer . The ‘L*’ value represents brightness and darkness, the ‘a*’ value represents greeness and redness as the value increases from negative to positive, and ‘b*’ represents blueness and yellowness. The ‘C*’ value represents chroma, calculated according to C* = 1/2(a*2+b*2). The hue angle (H) was calculated according to the following equation: H = arctan (b*/a*) For flower color evaluation, color components of the CIE −1. The elution program followed the procedure described by Wu and Prior Pigments were quantified by high-performance liquid chromatography (HPLC). One hundred milligrams of petals were homogenized with a mortar and pestle in liquid nitrogen, then 1 ml of extraction solution  was added and the mixture was stored overnight at 4°C in the dark. The mixture was then centrifuged at 4°C at 12,000×g for 15 min, and the supernatants were filtered through a 0.22 µm Millipore filter . Anthocyanins were identified and quantified using an HPLC1100-DAD system . Detection was performed at 520 nm for anthocyanins, 350 nm for flavonoids. A NUCLEODURH C18 column (250 mm64.66 mm)  operating at 25°C was used for separation and compounds were eluted in a mobile phase consisting of solvent A, (trifluoroacetic acid: formic acid: water [0.1∶2∶97.9]) and solvent B (trifluoroacetic acid: formic acid: acetonitrile: water [0.1∶2∶35∶62.9]) at a flow rate of 0.8 ml minMcCHS, McF3H, McF3′H, McDFR, McANS, McUFGT, McMYB1, McMYB2, McMYB3, McMYB4, McMYB5, McMYB6, McMYB7, McMYB10, McMYB14 were analyzed using quantitative real-time PCR (qRT-PCR) with SYBR Green qPCR Mix  and a Bio-Rad CFX96 Real-Time PCR System , according to the manufacturers’ instructions. The expression levels of NtCHS, NtF3H, NtF3′H, NtDFR, NtANS and NtUFGT were analyzed using the same techniques. Primers are designed by NCBI Primer BLAST and listed in Malus ribosomal 18S rRNA gene (DQ341382) was used as a reference gene for the target genes McCHS, McF3H, McF3′H, McDFR, McANS, and McUFGT, McMYB1, McMYB2, McMYB3, McMYB4, McMYB5, McMYB6, McMYB7, McMYB10, McMYB14; NtActin (GQ339768) was used as a reference gene for the target genes NtCHS, NtF3H, NtF3′H, NtDFR, NtANS, NtUFGT. The resulting cDNA samples for reverse transcription were serially diluted . Real-time RT-PCR analysis was carried out in a total volume of 20 µl, containing 9 µl of 2×SYBR Green qPCR Mix , 0.1 µM of each specific primer, and 100 ng of template cDNA. The reaction mixtures were heated to 95°C for 30 s, followed by 39 cycles at 95°C for 10 s, 59°C for 15 s, and 72°C for 30 s. A melting curve was generated for each sample at the end of each run to ensure the purity of the amplified products. Standard dilution curves were performed for each gene fragment, and all data were normalized to the level of the reference gene transcript. Primers for real-time experiments were designed using primer premier v5.0 software with forward and reverse primers corresponding to two different exons.Total RNA was extracted from flowers, leaves and fruit flesh and pericarp using an RNA Extract Kit , according to the manufacturer's instructions. DNase I  was added to remove genomic DNA and the samples were subjected to cDNA synthesis using the Access RT-PCR System  according to the manufacturer's instructions. The expression levels of CHS and aligned with translated genome reference sequences and published CHS protein sequences from other species with DNAMAN 5.2.2. A phylogenetic tree was produced using MEGA version 5.10 Protein consensus sequences were determined from the coding sequences of McCHS coding sequence was amplified by PCR with the McCHS-F and McCHS-R primers, using cDNA from ‘Royalty’ petals as a template. The primers for McCHS amplification contained SpeI and KpnI restriction enzyme sites, which were used to clone the coding region of the McCHS gene into the pBI121 vector. This was then used to transform Agrobacterium strain LBA4404 which in turn was used to transform tobacco (N. benthamiana) Wisconsin 38 using the leaf disk method The entire Statistical analysis and graphing was carried out using the OriginPro 8 statistical software . Microsoft Office PowerPoint 2003 was used for artwork. Error bars represents mean ± SE of three replicate reactions.Figure S1The phenotypes of different organs of the three Malus crabapple.(DOC)Click here for additional data file.Table S1Sequences of primers and relevant accession numbers.(DOC)Click here for additional data file.