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The formation and maintenance of sharp boundaries between groups of cells play a vital role during embryonic development as they serve to compartmentalize cells with similar fates. Some of these boundaries also act as organizers, with the ability to induce specific cell fates and morphogenesis in the surrounding cells. The midbrain-hindbrain boundary (MHB) is such an organizer: it acts as a lineage restriction boundary to prevent the intermingling of cells with different developmental fates. However, the mechanisms underlying the lineage restriction process remain unclear. Here, using novel fluorescent knock-in reporters, live imaging, Cre/lox-mediated lineage tracing, atomic force microscopy-based cell adhesion assays and mutant analysis, we analyze the process of lineage restriction at the MHB and provide mechanistic details. Specifically, we show that lineage restriction occurs by the end of gastrulation, and that the subsequent formation of sharp gene expression boundaries in the developing MHB occur through complementary mechanisms, i.e. cell-fate plasticity and cell sorting. Furthermore, we show that cell sorting at the MHB involves differential adhesion among midbrain and hindbrain cells that is mediated by N-cadherin and Eph-ephrin signaling., Competing Interests: Competing interestsThe 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., (© 2020. Published by The Company of Biologists Ltd.)

Neuropilin 1 (NRP1) is a receptor for class 3 semaphorins and vascular endothelial growth factor (VEGF) A and is essential for cardiovascular development. Biochemical evidence supports a model for NRP1 function in which VEGF binding induces complex formation between NRP1 and VEGFR2 to enhance endothelial VEGF signalling. However, the relevance of VEGF binding to NRP1 for angiogenesis in vivo has not yet been examined. We therefore generated knock-in mice expressing Nrp1 with a mutation of tyrosine (Y) 297 in the VEGF binding pocket of the NRP1 b1 domain, as this residue was previously shown to be important for high affinity VEGF binding and NRP1-VEGFR2 complex formation. Unexpectedly, this targeting strategy also severely reduced NRP1 expression and therefore generated a NRP1 hypomorph. Despite the loss of VEGF binding and attenuated NRP1 expression, homozygous Nrp1(Y297A/Y297A) mice were born at normal Mendelian ratios, arguing against NRP1 functioning exclusively as a VEGF164 receptor in embryonic angiogenesis. By overcoming the mid-gestation lethality of full Nrp1-null mice, homozygous Nrp1(Y297A/Y297A) mice revealed essential roles for NRP1 in postnatal angiogenesis and arteriogenesis in the heart and retina, pathological neovascularisation of the retina and angiogenesis-dependent tumour growth.

Experiments in cultured cells have implicated the molecular switch Rac in a wide variety of cellular functions. Here we demonstrate that the simultaneous disruption of two negative regulators of Rac, Abr and Bcr, in mice leads to specific abnormalities in postnatal cerebellar development. Mutants exhibit granule cell ectopia concomitant with foliation defects. We provide evidence that this phenotype is causally related to functional and structural abnormalities of glial cells. Bergmann glial processes are abnormal and GFAP-positive astroglia were aberrantly present on the pial surface. Older Abr;Bcr-deficient mice show spontaneous mid-brain glial hypertrophy, which can further be markedly enhanced by kainic acid. Double null mutant astroglia are hyper-responsive to stimulation with epidermal growth factor and lipopolysaccharide and exhibit constitutively increased phosphorylation of p38 mitogen-activated protein kinase, which is regulated by Rac. These combined data demonstrate a prominent role for Abr and Bcr in the regulation of glial cell morphology and reactivity, and consequently in granule cell migration during postnatal cerebellar development in mammals.

The developing vertebrate hindbrain is transiently subdivided along the anterior-posterior axis into metameric units, called rhombomeres (r). These segments constitute units of lineage restriction and display specific gene expression patterns. The transcription factor gene Krox-20 is restricted to r3 and r5, and is required for the development of these rhombomeres. We present evidence that Krox-20 transcriptional activity is under the control of a negative feedback mechanism in the hindbrain. This regulatory loop involves two closely related proteins, Nabl and Nab2, previously identified as antagonists of Krox-20 transcriptional activity in cultured cells. Here we show that in the mouse hindbrain, Nab1 and Nab2 recapitulate the Krox-20 expression pattern and that their expression is dependent on Krox-20 function. Furthermore, misexpression of Nab1 or Nab2 in zebrafish embryos leads to alterations in the expression patterns of several hindbrain markers, consistent with an inhibition of Krox-20 activity. Taken together, these data indicate that Krox-20 positively regulates the expression of its own antagonists and raise the possibility that this negative feedback regulatory loop may play a role in the control of hindbrain development.

The analysis of mice mutant for both Hoxa1 and Hoxb1 suggests that these two genes function together to pattern the hindbrain. Separately, mutations in Hoxa1 and Hoxb1 have profoundly different effects on hindbrain development. Hoxa1 mutations disrupt the rhombomeric organization of the hindbrain, whereas Hoxb1 mutations do not alter the rhombomeric pattern, but instead influence the fate of cells originating in rhombomere 4. We suggest that these differences are not the consequences of different functional roles for these gene products, but rather reflect differences in the kinetics of Hoxa1 and Hoxb1 gene expression. In strong support of the idea that Hoxa1 and Hoxb1 have overlapping functions, Hoxa1/Hoxb1 double mutant homozygotes exhibit a plethora of defects either not seen, or seen only in a very mild form, in mice mutant for only Hoxa1 or Hoxb1. Examples include: the loss of both rhombomeres 4 and 5, the selective loss of the 2(nd) branchial arch, and the loss of most, but not all, 2(nd) branchial arch-derived tissues. We suggest that the early role for both of these genes in hindbrain development is specification of rhombomere identities and that the aberrant development of the hindbrain in Hoxa1/Hoxb1 double mutants proceeds through two phases, the misspecification of rhombomeres within the hindbrain, followed subsequently by size regulation of the misspecified hindbrain through induction of apoptosis.